Protocol to measure apoptosis-associated speck-like protein containing a CARD specks in human cerebrospinal fluid via imaging flow cytometry

Summary

Apoptosis-associated speck-like protein containing a c-terminal caspase activation and recruitment domain (ASC) specks are elevated in the cerebrospinal fluid (CSF) of Alzheimer’s disease and related dementias (AD/ADRDs) patients. Here, we present a flow cytometry protocol to quantify ASC specks. We describe steps for fluorescently labeling ASC specks using antibody technology, visualizing with imaging flow cytometry, and gating based on physical characteristics. CSF ASC specks levels positively correlate with phosphorylated tau (Thr181) and negatively correlate with amyloid β ratio (42/40), thus serving as a neuroinflammatory biomarker for diagnosing AD/ADRDs.

For complete details on the use and execution of this protocol, please refer to Jiang et al.¹

Graphical abstract

Highlights

• •Purification of ASC specks from ASC-cerulean inflammasome reporter macrophages
• •Quantification of ASC specks in human cerebrospinal fluid with flow cytometry
• •Purified ASC specks and ASC knockout tissue are used to validate the methodology
• •ASC specks correlate with phosphorylated tau (T181) and amyloid β ratio in the CSF

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Apoptosis-associated speck-like protein containing a c-terminal caspase activation and recruitment domain (ASC) specks are elevated in the cerebrospinal fluid (CSF) of Alzheimer’s disease and related dementias (AD/ADRDs) patients. Here, we present a flow cytometry protocol to quantify ASC specks. We describe steps for fluorescently labeling ASC specks using antibody technology, visualizing with imaging flow cytometry, and gating based on physical characteristics. CSF ASC specks levels positively correlate with phosphorylated tau (Thr181) and negatively correlate with amyloid β ratio (42/40), thus serving as a neuroinflammatory biomarker for diagnosing AD/ADRDs.

Before you begin

The protocol below describes a novel method designed to visualize and quantify ASC specks in human cerebral spinal fluid (CSF) samples with imaging flow cytometry (Figure 1). These CSF samples were subjected to other assays that are not described in the protocol. These kits include the V-PLEX Plus Aβ Peptide Panel 1 (4G8) Kit (Meso Scale Discovery, K15199G-2) and Innotest Phospho-Tau Kit (181P; Fujirebio, 81582). The protocol for those assays can be found in the kits provided by the vendors. ASC specks have also been investigated in the following types of samples in the context of Alzheimer’s disease and related dementias (ADRDs); mouse brain homogenates,² cell culture media, human serum,³ human brain sections,⁴ human peripheral blood mononuclear cells (PBMCs),⁵ and CSF of small number of patients with tauopathies.¹ Note that while this protocol can be modified, the methodology described in this manuscript heavily relies on an imaging flow-cytometer. The high-resolution images acquired through the two-camera system are necessary to determine the size of the ASC specks since they are nanoparticles. The sensitivity of the imaging flow-cytometer is critical, and the described method has been optimized with the ImageStream X Mark II. This flow-cytometer has the sensitivity to detect particles smaller than 400–500 nm, which is the limit of detection for the photomultiplier tube of traditional flow cytometers.⁶ While there are other commercially available imaging flow-cytometers, it is recommended that any specifications be compared to the ImageStream X Mark II before use. See materials and equipment for ImageStream X Mark II specifications.

Figure 1.

Workflow required to visualize ASC specks

(A) Human CSF is collected through a lumbar puncture and aliquoted.

(B) The CSF is then subjected to MSD and Fujirebio.

(C) Another aliquot of the CSF sample is used for imaging flow cytometry.

(D) The CSF is centrifuged and incubated with goat serum.

(E and F) The samples are then incubated with primary rabbit ASC antibody (Adipogen) and washed with 10% goat serum.

(G) The ASC specks are then incubated with a fluorescent secondary antibody (Alexa Flour 647).

(H) The ASC specks are imaged through flow cytometry and size-based gating will exclude monomeric ASC-antibody complex.

(I) A specific gating strategy is used to quantify the number of assembled ASC specks in CSF samples.

Institutional permissions

All experiments performed with human cerebrospinal fluid (CSF) samples were approved by the University of New Mexico Institutional Review Board (IRB) protocol 04-294. All investigations that involved samples obtained from mice were approved by the University of New Mexico IACUC protocol 21-201179-HSC.

Prepare reagents for ASC-cerulean cell culture and inflammatory stimulation

Timing: 1 h

This section describes how to prepare reagents for in-vitro experiments that stimulate the release of ASC specks in ASC-cerulean macrophages described in the step-by-step method details section. The ASC specks are used as a positive control in the imaging flow-cytometer.

• 1.
  Prepare 250 mL of media for ASC-cerulean experiments.

  Note: More information related to these cells can be found in Stutz et al.⁷

  • a.
    Open the biosafety cabinet and wait a minimum of 30 min before use.
  • b.
    Open the Vacuum Filter/Storage Bottle System (Corning, 431096).
  • c.
    Use a 25 mL serological pipette to place 25 mL of 100% Heat Inactivated Fetal Bovine Serum (HI-FBS; Thermo Fisher Scientific, 10082147) over the filter.
  • d.
    Use a 5 mL serological pipette to add 2.50 mL Penicillin-Streptomycin (5,000 U/mL; Thermo Fisher Scientific, 15070063) over the filter.
  • e.
    Use a 50 mL serological pipette to add 222.50 mL Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (Cytiva, SH30243.02) over the filter.

    Note: Addition of media will bring the solution up to a final volume of 250 mL.

  • f.
    Attach the Vacuum Filter/Storage Bottle System to the vacuum system of the biosafety cabinet.
  • g.
    Turn on the vacuum in the biosafety cabinet.
  • h.
    Allow all the liquid to pass through the filter.
  • i.
    Turn the vacuum off.
  • j.
    Remove the filter top from the bottle.
  • k.
    Immediately place the bottle top onto the container.
  • l.
    Aliquot the media into 50 mL aliquots.
  • m.
    Store the media aliquots at 4°C until use.
• 2.
  Resuspend the lipopolysaccharide (LPS) from E. coli O111:B4.

  • a.
    Open the biosafety cabinet.

    CRITICAL: Wait a minimum of 30 min before use.

  • b.
    Open the 10 mg LPS vial (Millipore Sigma, L2880-10 MG).
  • c.
    Add 10 mL of sterile 1× PBS (Thermo Fisher Scientific, 10010023) to the vial slowly with a 10 mL serological pipette.

    Note: This will produce a final concentration of 1 mg/mL.

  • d.
    Mix the bottle well by gently swirling it.
  • e.
    Gently vortex the mixture.
  • f.
    Aliquot the LPS for future use.
  • g.
    Store LPS at −20°C for long term storage.
• 3.
  Prepare nigericin.

  • a.
    Open the biosafety cabinet and wait a minimum of 30 min before use.
  • b.
    Open the 10 mg vial of Nigericin sodium salt (InvivoGen, 28643-80-3).
  • c.
    Slowly add 2 mL of sterile 100% ethanol (VWR, 89125-172) to the vial with a 5 mL serological pipette.

    Note: This will produce a final stock concentration of 5 mg/mL.

  • d.
    Mix the bottle well by gently swirling it.
  • e.
    Gently vortex the mixture.
  • f.
    Aliquot the nigericin for future use.
  • g.
    Store nigericin at −20°C for long term storage.

Prepare reagents for ASC speck isolation

Timing: 2 h

In this section, reagents are prepared to purify the ASC specks released by ASC-cerulean macrophages in-vitro as described in the step-by-step method details section. The purified ASC specks may be used as a positive control in the imaging flow-cytometer.

• 4.
  Prepare Buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 320 mM sucrose; lysis buffer).

  • a.
    Pour 35–40 mL Milli-Q (MQ) water into a 50 mL beaker.
  • b.
    Add a stir bar to the water.
  • c.
    Weigh 238.30 mg of HEPES-2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid free acid, high purity HEPES salt (VWR, 0511-250G) and add it to the water.
  • d.
    Weigh 15.25 mg of hydrous magnesium chloride (MgCl₂; Millipore Sigma; M8266-100G) salt and add it to the water.
  • e.
    Weigh 20.80 mg of Ethylenediaminetetraacetic acid tetrasodium salt dihydrate salt (EDTA; Millipore Sigma, E6511-100G) and add it to the water.

    CRITICAL: Check the molecular weight of EDTA before use.

  • f.
    Weigh 19.0 mg of Ethylene glycol-bis (2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA; Millipore Sigma, E3889-10G) salt and add it to the water.
  • g.
    Weigh 37.30 mg of potassium chloride (KCl); Millipore Sigma, P3911-500G) and add it to the water.
  • h.
    Weigh 5.48 g Sucrose (Millipore Sigma, S0389-500G) and add it to the water.
  • i.
    Mix the solution well for a minimum of 15 min on a stir plate.
  • j.
    Use a pH meter to determine the pH of the buffer once the salt is in solution.
  • k.
    Add potassium hydroxide (KOH) or hydrochloric acid (HCl) as necessary to adjust the pH of the mixture to 7.5.
  • l.
    Use a 50 mL graduated cylinder to bring the solution up to a final volume of 50 mL with Milli-Q (MQ) water.
  • m.
    Store the buffer at 4°C until use.
• 5.
  Prepare 10 mM phenylmethylsulfonyl fluoride (PMSF; Millipore Sigma, 10837091001).

  • a.
    Weigh 1.74 mg of PMSF salt.
  • b.
    Add the salt to a 1.50 mL Eppendorf tube.
  • c.
    Slowly add 1 mL 100% ethanol with 1 mL pipette directly to the tube.

    Note: This will produce a final concentration of 1.74 mg/mL (10 mM).

  • d.
    Gently vortex the solution.
  • e.
    Store the PMSF at 4°C until use.

    CRITICAL: Only use the PMSF immediately before isolating ASC specks.

• 6.
  Prepare 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate hydrate (CHAPS) buffer (20 mM HEPES-KOH, 5 mM MgCl₂, 0.5 mM EGTA, 0.1 mM PMSF, 0.1% CHAPS).

  • a.
    Add 75 mL Milli-Q (MQ) water to 100 mL beaker.
  • b.
    Add a stir bar.
  • c.
    Weigh 476.60 mg HEPES salt and add it to the water.
  • d.
    Weigh 101.65 mg MgCl₂ hydrous salt and add it to the water.
  • e.
    Weigh 19 mg EGTA salt and add it to the water.
  • f.
    Weigh 100 mg CHAPS (Thermo Fisher Scientific, 28300) and add it to the water.
  • g.
    Mix the buffer well for a minimum of 15 min with a stir plate until all salts are in solution.
  • h.
    Use a pH meter and potassium hydroxide (KOH) to bring the pH of the buffer to 7.5.
  • i.
    Use a 100 mL graduated cylinder to bring the solution up to a final volume of 100 mL with Milli-Q (MQ) water.
  • j.
    Store the buffer at 4°C until use.
  • k.
    Use a 200 μL pipette tip to transfer 50 μL of 10 mM phenylmethylsulfonyl fluoride (PMSF; Millipore Sigma, 10837091001) to a 50 mL aliquot of CHAPS buffer.

CRITICAL: Only add the PMSF immediately before isolating ASC specks.

Note: Add fresh PMSF for every isolation if the CHAPS is not used frequently.

• 7.
  Prepare 2.5 M sucrose solution.

  • a.
    Add 10 mL of Milli-Q (MQ) water to a 25 mL beaker.
  • b.
    Add a stir bar.
  • c.
    Weigh 1.28 g sucrose.
  • d.
    Slowly add sucrose to the beaker of water until it dissolves well.
  • e.
    Mix the buffer well for a minimum of 15 min with a stir plate until all salts are in solution.
  • f.
    Bring the solution up to a final volume of 15 mL with a 25 mL graduated cylinder.
  • g.
    Gently vortex the mixture.
  • h.
    Store the sucrose at 4°C until use.
• 8.
  Prepare 40% Percoll.

  • a.
    Use a serological 25 mL pipette to transfer 28 mL of MQ water to a 50 mL conical tube.
  • b.
    Use a 2 mL serological pipette to transfer 2 mL of Percoll solution to the water (Millipore Sigma, 17089102).
  • c.
    Mix the conical well by gently swirling.
  • d.
    Gently vortex the mixture.
  • e.
    Store the solution at 4°C until use.

Prepare reagents for flow cytometry (24 samples)

Timing: 3 weeks

In this section, reagents are prepared for the fluorescent staining of ASC specks in human samples described in the step-by-step method details section.

• 9.
  Optimize primary antibody (rabbit anti-ASC antibody; Adipogen, AG-25B-0006-C100) with the appropriate positive and negative controls described below.

  • a.
    Purify ASC specks in the procedure described in the step-by-step method details section.
  • b.
    Collect cortical brain protein lysate from ASC knockout mice.

    Note: Unstimulated ASC-cerulean macrophages may also be used as a negative control.

  • c.
    Fluorescently stain the positive and negative control samples.

    • i.
      Transfer 10 μL purified ASC specks to a 1.5 mL Eppendorf tube (positive control)
    • ii.
      Transfer 10 μL ASC knockout homogenized brain tissue to a 1.5 mL Eppendorf tube (negative control).
    • iii.
      Follow Steps 59–67 in as described in the step-by-step method details section.
    • iv.
      Compare two dilutions of primary antibody with the positive and negative controls.

      Note: This experiment determined that 1:100 was the best primary antibody concentration when compared to 1:1000 (Figure 2).

    • v.
      Follow Steps 69–78 in as described in the step-by-step method details section to complete the fluorescent staining of the samples.
  • d.
    Visualize the stained samples on the imaging flow-cytometer as described in the step-by-step method details section.
  • e.
    Use the gating strategy described in the quantification and statistical analysis section to analyze the images.
  • f.
    Determine the number of ASC specks in each sample.

    Note: The optimal antibody concentration is one that the number of ASC specks counted in the purified ASC speck sample is well above the negative control sample (Figure 2).

• 10.
  Prepare 1 L 1× PBS (phosphate buffer saline; calcium and magnesium free).

  • a.
    Measure 100 mL 10× PBS with a 1 L graduated cylinder (Thermo Fisher Scientific, 70011-044).
  • b.
    Bring the solution up to volume with 900 mL of Milli-Q water.
  • c.
    Mix the solution well.
• 11.
  Prepare blocking solution (10% normal goat serum in 1× PBS).

  • a.
    Measure 27 mL of 1× PBS with a 50 mL graduated cylinder or appropriate conical.
  • b.
    Use a 5 mL serological pipette to add 3 mL of 100% normal goat serum (MP Bio, 191356) directly to the 1× PBS.
• 12.
  Prepare primary antibody (Adipogen rabbit anti-ASC antibody, AG-25B-0006-C100).

  CRITICAL: Prepare a 1:100 dilution; 50 μL per sample.

  • a.
    Use a 5 mL serological pipette to transfer 1.30 mL of blocking solution to a 2 mL Eppendorf tube.
  • b.
    Use a 20 μL pipette tip to pipette 13 μL of the primary antibody directly into the 2 mL Eppendorf tube.
• 13.
  Prepare fluorescent goat anti-rabbit secondary antibody (Thermo Fisher Scientific Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647, A-21245).

  CRITICAL: Prepare a 1:500 dilution; 250 μL per sample.

  • a.
    Transfer 6.20 mL of blocking solution to a 15 mL conical centrifuge tube with a 10 mL serological pipette.
  • b.
    Use a 20 μL pipette tip to pipette 12.40 μL of the secondary antibody into the conical.

    CRITICAL: Keep antibody cocktail away from light since the secondary antibody is fluorescent.

CRITICAL: Mix all solutions well before use.

Figure 2.

ASC-primary antibody titer

(A and B) (A) ASC-cerulean macrophages were grown in cell culture dishes and subjected to inflammatory agents. (B) ASC specks were then purified from the cell lysates.

(C–F) After treatment, the cells were lysed and subjected to a series of steps including centrifugation and washes with 1× CHAPS.

(G) A 40% Percoll gradient was then used to further purify the ASC specks.

(H–J) The purified ASC specks were then stained with primary antibody and subjected to imaging flow cytometry. The number of ASC specks detected at the 1:1,000 dilution (n = 3) is lower than the number detected with the 1:100 dilution (n = 3). Values are reported as mean ± SEM, using an unpaired Student’s t-test; p = 0.0544.

Set-up fluid bay reagents in the imaging-flow cytometer instrument

Timing: 15 min

This section describes how to prepare reagents needed for the fluid bay of the imaging flow-cytometer.

• 14.
  Prepare 10% bleach (1 L).

  • a.
    Pour 100 mL 100% bleach (Thomas Scientific, 1201R80) into a 1 L graduated cylinder.
  • b.
    Transfer 900 mL of Milli-Q water into the graduated cylinder to bring the solution up to volume.
  • c.
    Mix well.
  • d.
    Store the 10% bleach solution in a bleach safe storage container until use.

Note: Keep the solution away from areas of the laboratory that must remain free of bleach such as cell culture facilities. Dispose of any bleach waste according to institutional and state guidelines.

• 15.
  Prepare 70% ethanol (1 L).

  • a.
    Measure 700 mL ethanol with a 1 L graduate cylinder.
  • b.
    Bring the solution up to volume by adding 300 mL Milli-Q water to the graduated cylinder.
  • c.
    Mix well.

Note: See Alternatives if isopropanol is preferred.

• 16.Open the fluid bay of the imaging flow-cytometer (ImageStream X Mark II).
• 17.
  Refill empty containers in the fluid bay.

  • a.
    Carefully detach the plastic bottle cap from the appropriate reagent bottle.
  • b.
    Detach the receptacle from the machine.
  • c.
    Refill the empty receptacle with the appropriate reagent.

    Note: For example, refill the bleach container if it is empty.

  • d.
    Reattach the receptacle to the machine.
  • e.
    Repeat Step 17 as appropriate for every empty container.
• 18.
  Empty the liquid waste container.

  • a.
    Carefully unscrew the metallic attachments that connect the waste receptacle to the flow-cytometer.
  • b.
    Once the metallic attachments are unscrewed, detach the receptacle from the machine.
  • c.
    Dispose of the waste according to institutional standards.
  • d.
    Place the waste receptacle back in the machine.
  • e.
    Use the metallic attachments to screw the receptacle back into the flow-cytometer.
• 19.
  Once any reagents have been replaced, verify that the fluid bay contains the following before beginning any experiment:

  • a.
    Speed Beads (Cytek, 400041).
  • b.
    70% Ethanol (200 proof and undenatured; VWR, 89125-172).
  • c.
    Beckman Coulter Coulter Clenz (Beckman Coulter, 8546929).
  • d.
    10% Bleach (Thomas Scientific, 1201R80).
  • e.
    1× PBS (Thermo Fisher, 70011044).

Note: Fresh reagents may be added to the fluid bay a day before the experiment.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal anti-ASC (1:100)	AdipoGen Life Sciences	Cat# AG-25B-0006-C100; RRID: AB_2885200
Goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647 (1:500).	Thermo Fisher Scientific	Cat# A-21245; RRID: AB_2535813
Biological samples
Human CSF (refer to Table S1 for additional details).	UNM Center for Memory and Aging	N/A
Chemicals, peptides, and recombinant proteins
LPS	Sigma	Cat# L2880-10MG
Nigericin	InvivoGen	Cat# 28643-80-3
Critical commercial assays
V-PLEX Plus Aβ Peptide Panel 1 (4G8) Kit	Meso Scale Discovery	Cat# K15199G-2
INNOTEST phospho-tau (181P)	Fujirebio	Cat# 81581
Experimental models: Cell lines
ASC-mCerulean macrophages	Gift from Dr. Eicke Latz	Stutz et al.7
Experimental models: Organisms/strains
ASC−/− female mice (3.5 months old)	Gift from Dr. Vishwa Dixit (Genentech)	Mariathasan et al.8
Software and algorithms
Amnis INSPIRE	Cytek Biosciences	https://cytekbio.com/pages/imagestream
Amnis IDEAS		Cytek Biosciences
Prism	GraphPad	https://www.graphpad.com/scientific-software/prism/; RRID: SCR_002798
Other
DMEM/high glucose with L-glutamine, sodium pyruvate	Cytiva	Cat# 30243.02
Penicillin-Streptomycin (5,000 U/mL)	Thermo Fisher Scientific	Cat# 15070063
Fetal bovine serum, certified, heat inactivated, United States	Thermo Fisher Scientific	Cat# 10082147
Normal goat serum	MP Biomedicals	Cat# 191356
Phenylmethylsulfonyl fluoride (PMSF)	MilliporeSigma	Cat# 10837091001
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS)	MilliporeSigma	Cat# 226947-1G
Sucrose for molecular biology, ≥99.5% (GC)	MilliporeSigma	Cat# S0389-500G
Ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA)	MilliporeSigma	Cat# E6511-100G
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA)	MilliporeSigma	Cat# E3889-10G
Potassium chloride (KCl)	MilliporeSigma	Cat# P3911-500G
Magnesium chloride (MgCl2)	MilliporeSigma	Cat# M8266-100G
HEPES-2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid free acid, high purity	VWR	Cat# 0511-250G
Hydrochloric acid solution	MilliporeSigma	Cat# H9892
Trypsin-EDTA solution	MilliporeSigma	Cat# T4174
Corning 250 mL vacuum filter/storage bottle system, 0.22 μm pore 19.6 cm2 PES membrane, sterile, 12/case	Corning	Cat# 431096
Corning T-75 U-shaped cell culture flasks	Fisher Scientific	Cat# 07-202-000
Corning tissue culture-treated culture dishes	MilliporeSigma	Cat# CLS430165
Corning cell lifter	Corning	Cat# 3008
Clorox liquid bleach	Thomas Scientific	Cat# 1201R80
200 proof pure ethanol	VWR	Cat# 89125-172
PBS (10×), pH 7.4	Thermo Fisher Scientific	Cat# 70011044
PBS (1×), pH 7.4	Thermo Fisher Scientific	Cat# 10010-023
Self-stand screw-cap vials	USA Scientific	Cat# 1405-9710
Amnis SpeedBead kit for ImageStreamX system, ISX400041	Cytek Biosciences	Cat# CN-0440-01
Coulter Clenz agent (500 mL)	Beckman Coulter	Cat# 8546929
Pierce BCA Protein Assay Kit, 1,000 mL	Thermo Scientific	Cat# 23225
Tissue protein extraction reagent	Thermo Fisher Scientific	Cat# 78510
PageRuler prestained protein ladder, 10–180 kDa	Thermo Fisher Scientific	Cat# 26616
Invitrolon PVDF/filter paper sandwiches, 0.45 μm, 8.3 × 7.3 cm (for mini gels)	Thermo Fisher Scientific	Cat# LC2005
Mini-PROTEAN TGX gels	Bio-Rad	Cat# 4561093
Blotting-grade blocker	Bio-Rad	Cat# 1706404
4× Laemmli protein sample buffer for SDS-PAGE	Bio-Rad	Cat# 610747
Protease inhibitor cocktail	MilliporeSigma	Cat# P8340
Phosphatase inhibitor cocktail	MilliporeSigma	Cat# P5726
Percoll	MilliporeSigma	Cat# GE17-0891-02
Roto-Mini rotator	Benchmark Scientific	Cat# R2020∗
Prism R refrigerated microcentrifuge	Labnet	Cat# Z723762
ImageStreamX Mark II (AMNIS)	Cytek	Cat# 100220

Materials and equipment

ASC cerulean macrophage cell culture

ASC-cerulean cell culture media

	Final concentration	Amount
Penicillin-Streptomycin (5,000 U/mL)	1×	2.50 mL
Fetal Bovine Serum	10%	25 mL
Dulbecco’s Modified Eagle’s Medium-high glucose	N/A	222.50 mL
Total		250 mL

Keep stable at 4°C.

1× Trypsin/EDTA

	Final concentration	Amount
10× Trypsin/EDTA	1×	300 μL
1× PBS	1×	2.70 mL
Total		3 mL

Store at −20°C for long-term storage. After an aliquot is thawed out keep it refrigerated at 4°C.

LPS

	Final concentration	Amount
LPS salt	1 mg/mL	10 mg
1× PBS	1×	10 mL
Total		10 mL

Keep stable at 4°C or on ice until use. Store at −20°C for long-term storage.

Nigericin

	Final concentration	Amount
Nigericin salt	5 mg/mL	10 mg
Ethanol	100%	2 mL
Total		2 mL

Keep stable at 4°C or on ice until use. Store at −20°C for long-term storage.

ASC speck purification

Buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 320 mM sucrose)

	Final concentration	Amount
HEPES	20 mM	238.30 mg
KCl	10 mM	37.30 mg
MgCl2	1.5 mM	15.25 mg
EDTA	1 mM	20.80 mg
EGTA	1 mM	19 mg
Sucrose	320 mM	5.48 g
MQ Water	N/A	50 mL
Total		50 mL

Keep stable at 4°C or on ice until use.

PMSF

	Final concentration	Amount
PMSF	10 mM	1.74 mg
Ethanol	100%	1 mL
Total		1 mL

Keep stable at 4°C or on ice until use.

CHAPS Buffer: 20 mM HEPES-KOH, pH 7.5, 5 mM MgCl₂, 0.5 mM EGTA, 0.1 mM PMSF, 0.1% CHAPS

	Final concentration	Amount
HEPES	20 mM	276.60 mg
MgCl2	5 mM	101.65 mg
EGTA	0.5 mM	19 mg
CHAPS	0.1%	100 mg
MQ Water	N/A	100 mL
Total		100 mL

Keep stable at 4°C or on ice until use.

Add PMSF (1:1000 dilution of 10 mM stock) immediately before use.

2.5 M Sucrose

	Final concentration	Amount
Penicillin-Streptomycin (5,000 U/mL)	1×	2.50 mL
Fetal Bovine Serum	10%	25 mL
Total		25 mL

Keep stable at 4°C or on ice until use.

40% Percoll

	Final concentration	Amount
2.5 M Sucrose	10%	2 mL
Percoll	90%	18 mL
Total		20 mL

Keep stable at 15°C–25°C until use.

Note: The Eppendorf Centrifuge 5415 R was used for all centrifugation steps described in this protocol.

Fluorescent staining and imaging flow-cytometry

Note: The imaging flow-cytometer used to describe this method is the ImageStream X Mark II and has the following specifications: An upgraded 2 camera system that includes bright field, side scatter (SSC) and up to 10 fluorescent channels (Ch 1: 457/45, Ch 2: 528/65, Ch 3: 577/35, Ch 4: 610/30, Ch 5: 702/85, Ch 6: 762/35, Ch7: 457/45, Ch 8: 537/65, Ch9: 582/25, Ch 10: 610/30, Ch 11 702/85, Ch12: 762/35). The ImageStream X Mark II has an expanded 5 laser configuration that offers 405 nm, 488 nm, 561 nm, 642 nm, and 785 nm excitation.

CRITICAL: With a multi-mag upgrade, samples can be imaged at 20×, 40×, or 60× magnification with highest possible pixel resolutions at 1 × 1 μm² (at 20×), 0.5 × 0.5 μm² (at 40×), and 0.3 × 0.3 μm² (at 60×), respectively. 60× gives a core diameter of 7 μm while 40× and 20× have core diameters of 10 μm. Note that the system did not have the multi-mag option at the time of data acquisition and had a single magnification of 40×.

Blocking solution (10% goat serum)

Reagent	Final concentration	Amount
100% Normal goat serum (NGS)	10%	3 mL
PBS	1×	27 mL
Total	N/A	30 mL

Keep stable at 4°C or on ice until use. 100% NGS can be stored at −20°C for long-term storage.

Primary antibody (Anti-rabbit ASC polyclonal Antibody-AL177)

Reagent	Final concentration	Amount
Blocking solution	10% normal goat serum	1.29 mL
Primary antibody	1:100; 0.01 mg/mL	13 μL
Total	N/A	1.30 mL

Keep stable at 4°C or on ice until use.

Anti-ASC primary antibody can be stored at 4°C for short-term use, and at −20°C for long-term storage.

Secondary antibody (goat anti-rabbit Alexa Fluor 647)

Reagent	Final concentration	Amount
Blocking solution	10% normal goat serum	6.18 mL
Secondary antibody	1:500; 4 μg/mL	12.40 μL
Total	N/A	6.20 mL

Keep stable at 4°C or on ice until use.

Goat anti-rabbit Alexa Fluor 647 can be stored in the dark at 4°C.

1× PBS

Reagent	Final concentration	Amount
10× PBS	1×	100 mL
Milli-Q water	N/A	900 mL
Total	N/A	1 L

Store at 15°C–25°C.

10% Bleach

Reagent	Final concentration	Amount
100% Bleach	10%	100 mL
Milli-Q water	N/A	900 mL
Total	N/A	1 L

Store at 15°C–25°C.

CRITICAL: Make 10% bleach fresh daily and follow local guidelines related to the use, storage, and disposal of 100% bleach.

Note: All dilutions described in this section were done by taking the total volume of diluent and removing the volume of diluent to be replaced by the reagent(s) with a smaller volume.

Note: The Eppendorf Centrifuge 5415 R was used to centrifuge samples in all experiments.

Alternatives: To isolate purified ASC specks, immunoprecipitation may be conducted instead of the method recommended in this protocol.² Different reagents may also be utilized for the fluorescent staining of ASC specks in CSF samples. For example, any commercially available primary antibody that binds specifically to human ASC may label ASC specks. However, a titration must be conducted with the new primary antibody using proper controls to validate the modification. Such controls include purified ASC specks isolated from ASC-cerulean macrophages and cortical tissue from Pycard deficient (ASC knockout) mice (Figure 2).^1,8 Alternative blocking solutions may also be tested such as donkey serum and bovine serum albumin. Note that if a different blocking solution is used that the secondary antibody should be changed accordingly. If such experimental modifications are anticipated, the method should be further validated with controls as described above.^1,8 Proper controls and antibody titration will ensure that the ASC fluorescent signal observed in the CSF samples are due to specific antibody binding. Alternative reagents can be used for image acquisition depending on the type of imaging flow-cytometer that is used. Solutions stored in the fluid bay such as ethanol and bleach are not critical to the measurement of ASC specks and are used to maintain the instrument. Therefore, these reagents can be purchased from any generic supplier or even substituted. For example, 70% isopropanol may be used instead of ethanol. Note that isopropanol should be used if there are concerns that the sample may clog the flow-cytometer. We also recommend the use of alcohol consistent with institutional protocols and biological waste regulations. Speed Beads are another reagent related to image acquisition that may be modified. For example, Speed Beads of another size may be used in the flow-cytometer. Before using any new size of speed bead, consider that the gating parameters used in this protocol will need to be modified based on the size of the speed bead used. Speed Beads (Cytek, 400041) that are 1 μm were purchased as a size calibrator for this protocol. While the size of Speed Beads can be adjusted, they are not optional and are necessary for proper image acquisition. For example, they are needed for camera synchronization and auto focus. Speed Beads also run parallel to the cell stream, allowing the flowrate of the system to be calibrated. Lastly, they help determine if the tubing is clogged.

Step-by-step method details

ASC-cerulean macrophage cell culture

Timing: 2–3 weeks

This section describes how to grow ASC-cerulean macrophages. These cells must be thawed out and maintained to induce the release of ASC specks. The ASC specks isolated from these cells may later be used as a positive control in the imaging flow-cytometer.

• 1.
  Thaw out ASC-cerulean macrophages from cryopreservation.⁷

  • a.
    Open the biosafety cabinet.

    CRITICAL: Wait a minimum of 30 min before use.

  • b.
    Pre-warm a 50 mL aliquot of media at 37°C in the water bath for 15 min.

    CRITICAL: Add proper growth supplements that are not already in DMEM.

  • c.
    Use a 10 mL serological pipette to transfer 8 mL of fresh media to a 15 mL conical tube.
  • d.
    Transfer 10 mL of fresh media to a T-75 U-Shaped Cell Culture flask with a 10 mL serological pipette (Fisher Scientific, 07-202-000).

    CRITICAL: Place the flask in the CO₂ incubator at 37°C until use.

  • e.
    Quickly remove the cell vial containing ASC-cerulean macrophages from long term liquid nitrogen storage.
  • f.
    Place the vial in a water bath for 3–5 min or until the liquid in the vial is completely thawed out.
  • g.
    Slowly add 1 mL of media to the cell vial with a 5 mL serological pipette.
  • h.
    Mix well by gently triturating 3–5 times.
  • i.
    Place the cells directly into the 15 mL conical tube containing fresh media with the same serological pipette.
  • j.
    Add 1 mL of fresh media to the vial again with a 1 mL pipette tip to obtain any residual cells still in the vial.
  • k.
    Use a 1 mL pipette tip to transfer the residual cells directly to the 15 mL conical tube.
  • l.
    Centrifuge the cells at 500 g for 5 min
  • m.
    Remove the supernatant with a 10 mL serological pipette or vacuum system.
  • n.
    Use a 5 mL serological pipette to resuspend the cell pellet with 1 mL of fresh media.
  • o.
    Triturate the cell suspension 7–10 times.
  • p.
    Place the cell suspension into the T-75 flask containing media.

    Note: The flask should now have a final volume of 11 mL.

  • q.
    Store the cells in a CO₂ incubator at 37°C for 2–3 days.
• 2.
  Split ASC-cerulean macrophages.

  • a.
    Open the biosafety cabinet.

    CRITICAL: Wait a minimum of 30 min before use.

  • b.
    Check the flask(s) for confluency with a light microscope.

    Note: If flask is 80%–90% confluent, proceed to split the cells. See troubleshooting 1.

  • c.
    Prewarm a 50 mL aliquot of media at 37°C in a water bath.
  • d.
    Prewarm the Trypsin-EDTA solution (Millipore Sigma, T4174) between 15°C–25°C.
  • e.
    Transfer 10.50 mL of fresh media to 6 clean T-75 flasks with a 10 mL serological pipette.

    CRITICAL: Place them in the CO₂ incubator at 37°C until use.

  • f.
    Place the T-75 flask containing the cells in the biosafety cabinet.
  • g.
    Use a 10 mL serological pipette or vacuum system to completely remove the old media from the T-75 flask.
  • h.
    Add 10 mL of 1× PBS with a 10 mL serological pipette to wash the flask.
  • i.
    Remove the 1× PBS from the flask with a 10 mL serological pipette or vacuum system.
  • j.
    Add 2.70 mL of fresh 1× PBS to the flask with a 5 mL serological pipette.
  • k.
    Use a 1 mL pipette tip to directly add 300 mL 10× Trypsin/EDTA to the flask.

    Note: The flask should now have a volume of 3 mL.

  • l.
    Gently tap the flask multiple times.
  • m.
    Place the flask in the CO₂ incubator at 37° for 3–5 min.
  • n.
    Verify with a light microscope that the cells have lifted.
  • o.
    Once the cells have lifted, add 6 mL of fresh media to the flask to stop the trypsin with a 5 mL serological pipette.

    CRITICAL: Media must contain FBS to stop the enzymatic activity of the Trypsin/EDTA.

  • p.
    Triturate the cells 7–10 times with the same serological pipette.
  • q.
    Place the cells into a fresh 15 mL conical tube.
  • r.
    Centrifuge the cells for 5 min at 500 g.
  • s.
    Remove the supernatant with a 10 mL serological pipette or vacuum system.
  • t.
    Use a 5 mL serological pipette to resuspend the cell pellet with 5 mL fresh media.
  • u.
    Add 500 μL of the cell suspension to each flask with a 1 mL pipette tip (1:10 dilution).

    Note: The large number of flasks allows for the expansion of the cell line to increase the possible ASC speck yield.

  • v.
    Place the flasks in the CO₂ incubator at 37°C for 2–3 days.
• 3.
  Plate ASC-cerulean cells for ASC-isolation and enrichment.

  • a.
    Open the biosafety cabinet.

    CRITICAL: Wait a minimum of 30 min before use.

  • b.
    Check the flasks for confluency with a light microscope.

    Note: If the cells are 80%–100% confluent, place the flasks in the biosafety cabinet for plating.

  • c.
    Prewarm 150 mL of media at 37°C in the water bath.
  • d.
    Prewarm the 10× Trypsin/EDTA solution between 15°C–25°C.
  • e.
    Use a 5 mL serological pipette to transfer 5 mL of fresh media to 24 cell culture dishes (Millipore Sigma, CLS430165).
  • f.
    Use a 10 mL serological pipette to remove the media from each flask.
  • g.
    Wash each flask with 10 mL of 1× PBS using a 10 mL serological pipette.
  • h.
    Remove the 1× PBS from each flask with a 10 mL serological pipette or vacuum system.
  • i.
    Add 2.70 mL of fresh 1× PBS to each flask with a 5 mL serological pipette.
  • j.
    Add 300 μL 10× Trypsin/EDTA to each flask with a 1 mL pipette tip.

    Note: Each flask should now have a volume of 3 mL.

  • k.
    Gently tap the flasks.
  • l.
    Place the flasks in the CO₂ incubator for 3–5 min at 37°C.
  • m.
    Verify with a light microscope that the cells have fully lifted.
  • n.
    When the cells have completely lifted, transfer 6 mL of fresh media directly to each flask with a 10 mL serological pipette.
  • o.
    Triturate the cell suspension in each flask 7–10 times with a 10 mL serological pipette.
  • p.
    Use a 10 mL serological pipette to transfer the cell suspension from each flask to a clean 50 mL conical tube.
  • q.
    Centrifuge the cells for 5 min at 500 g.
  • r.
    Remove the supernatant with a 10 mL serological pipette or vacuum system.
  • s.
    Resuspend the cells in 12 mL of fresh media with a 5 mL serological pipette.
  • t.
    Triturate the cell suspension well.
  • u.
    Add 500 μL of cell suspension to each dish with a 1 mL pipette tip.

    Note: Each cell culture dish should now have a final volume of 5 mL.

  • v.
    Place the cell culture dishes in the CO₂ incubator at 37°C for 1–2 days.

ASC-cerulean macrophage inflammasome priming and activation

Timing: 6 h

This section describes how to stimulate the release of ASC specks in ASC-cerulean macrophages. LPS and nigericin are used to prime and activate the inflammasome. ASC specks are released by the cells in response to the inflammatory stimuli.

• 4.Open the biosafety cabinet.

CRITICAL: Wait a minimum of 30 min before use.

• 5.Check that the cells are confluent with a light microscope.

Note: If the cells are 80%–100% confluent, proceed to the next step. If they are not confluent, do not continue and refeed the cells instead.

• 6.Use a 10 pipette μL tip to directly add 1.25 μL of 1 mg/mL LPS to the 5 mL of old media in the cell culture dish.

Note: This results in a final concentration of 250 ng/mL.

• 7.Gently rock each cell culture dish back and forth to mix the LPS well.
• 8.Incubate the cells for 3 h at 37°C in a CO₂ incubator.
• 9.Use a 10 μL pipette tip to directly add 14.92 μL of 5 mg/mL nigericin directly to the cell culture dish.

CRITICAL: Do not remove the media. This is a 1:335 dilution of the 5 mg/mL stock and results in a final concentration of 20 μM.

• 10.Incubate the cells for 1 h at 37°C in a CO₂ incubator.
• 11.Check the cells with a light microscope to determine if most cells are in suspension or adherent. See troubleshooting 2.
• 12.Place the cell culture dishes on ice.
• 13.Remove the media from each cell culture dish with a 10 mL serological pipette.

CRITICAL: Do not dispose of the media. It is necessary for later steps.

• 14.Transfer the media to a 50 mL conical.

CRITICAL: Store the media temporarily on ice.

• 15.Use a 1 mL pipette tip to add 1 mL ice cold 1× PBS to each cell culture dish.
• 16.Scrape the cell culture dishes to take off the adherent cells.

Note: Use a cell lifter (Corning, 3008) for best results.

• 17.Draw the scraped cells up with a 1 mL pipette tip.
• 18.Place the scraped cells into a fresh 50 mL conical.

CRITICAL: Store it temporarily on ice.

• 19.Repeat Steps 15–18 two more times for a total of three 1× PBS washes.
• 20.Centrifuge both the cell lysate and media at 500 g for 5 min.

CRITICAL: Set the centrifuge to 4°C.

• 21.Remove the media from the media pellet.

Optional: Harvest media.

• 22.Store the media pellet at −80°C until use.

Note: This pellet contains extracellular ASC, which can be used as a positive control for the flow cytometry protocol.

• 23.Remove the supernatant from the cell pellet.
• 24.Store the cell pellet at −80°C.

Note: This pellet contains intracellular ASC, which can be used as a positive control for the flow cytometry protocol.

Isolation of ASC specks from ASC-cerulean macrophages

Timing: 3 h

This section describes how to purify ASC specks from the ASC-cerulean macrophages. The purified ASC specks can be used as a positive control for fluorescent staining and flow cytometry.

CRITICAL: Maintain the samples at 4°C with ice at all steps.

Optional: Store all pellets and supernatants for future use.

• 25.Turn on the centrifuge.

CRITICAL: Set the temperature of the centrifuge to 4°C.

• 26.Pipette PMSF stock solution (1:1000) directly into the CHAPS buffer.

Note: For example, add 50 μL of 10 mM PMSF to 50 mL of CHAPS buffer.

• 27.Take out the cell and media pellets from −80°C storage.
• 28.Use a 1 mL pipette tip to add 0.5 mL Buffer A (lysis buffer) to the pellets.

Note: Only add Buffer A to the pellet once it is thawed out.

• 29.Incubate the pellets in Buffer A for a minimum of 30 min on ice.
• 30.Use a 1 mL pipette tip to transfer the cell lysate to a 2 mL Eppendorf Centrifuge tube.
• 31.Centrifuge the lysate at 212 g for 10 min.
• 32.Use a 1 mL pipette tip to transfer the supernatant to a fresh 2 mL Eppendorf tube.
• 33.Add 0.5 mL of Buffer A to the supernatant with a 1 mL pipette tip.

Note: This will bring the volume of the cell suspension to 1 mL.

• 34.Transfer 1 mL CHAPS/PMSF buffer to the supernatant with a 1 mL pipette tip.
• 35.Centrifuge the sample at 2351 g for 10 min.
• 36.Remove the supernatant with a with a 1 mL pipette tip.
• 37.Use a 1 mL pipette tip to resuspend the crude pellet with 1 mL CHAPS/PMSF.
• 38.Transfer 1 mL 40% Percoll to a fresh 2 mL Eppendorf tube.
• 39.Slowly place the 1 mL of CHAPS/PMSF containing the resuspended pellet over the 40% Percoll layer.

CRITICAL: Use a transfer pipette for best results.

• 40.Centrifuge the mixture at 17136 g for 10 min.
• 41.Remove the entire supernatant with a 1 mL pipette tip.

Note: A pellet may not be visible at this step but proceed with removing the supernatant.

• 42.Add 1 mL CHAPS/PMSF to the pellet with a 1 mL pipette tip.
• 43.Centrifuge the resuspended pellet for 10 min at 17136 g.
• 44.Remove the supernatant with a 1 mL pipette tip.
• 45.Add 1 mL CHAPS/PMSF to resuspend the pellet with a 1 mL pipette tip.
• 46.Centrifuge the sample for 10 min at 17136 g.
• 47.Remove the supernatant with a 1 mL pipette tip.
• 48.Resuspend the ASC specks in 50 μL CHAPS for future use such as flow cytometry.

Optional: Conduct a Pierce BCA protein assay (Thermo Scientific, 23225) to estimate the yield. See troubleshooting 3.

• 49.Store the ASC specks at −80°C until use.

Storage of CSF from lumbar puncture

Timing: 4 h

This section describes how to properly collect and store human samples for long-term storage. This step is necessary to measure the amount of ASC specks in CSF obtained from individuals with ADRD.

CRITICAL: Do not use any CSF sample that may be contaminated with blood.

• 50.Collect 20–30 mL CSF at the L3/L4 position with a 22-gauge Sprotte spinal needle via gravity flow.⁹
• 51.Collect the initial 1–2 mL in a 15 mL polypropylene conical tube.
• 52.Collect an additional 20–30 mL of CSF into sterile 15 mL polypropylene tubes.
• 53.Keep the sample on ice.
• 54.Centrifuge the CSF briefly at 2000 g for 10 min at 4°C.

Note: This will pellet any cellular debris.

• 55.Transfer the CSF from both 15 mL conical tubes into a 50 mL conical tube.

CRITICAL: Leave the debris at the bottom of each 15 mL centrifuged tube.

• 56.Gently invert the 50 mL conical tube 3–4 times to mix the sample.
• 57.Aliquot the CSF for future experiments in 0.5 mL Self-Stand Screw-Cap Vials (USA Scientific, 1405-9710).
• 58.Store the samples at −80°C for long term storage.

Pause point

Note: Samples are stable for long-term storage at this temperature. Avoid multiple freeze/thaw cycles of the CSF samples.

Immunostaining of CSF sample

Timing: 8 h

In this section, the fluorescent staining of ASC specks in human CSF with antibody technology is described. The sample is blocked with goat serum and incubated with anti-rabbit ASC antibody. Assembled ASC specks are then visualized with a fluorescent secondary antibody.

CRITICAL: Once staining has begun, it MUST proceed without interruption. If additional fluorophores are used, consider the need of a single-color control for each fluorophore to set up the compensation matrix.

• 59.Thaw out CSF samples on ice.

Optional: Thaw out ASC specks isolated from ASC-cerulean macrophages. This sample can serve as a positive control.

• 60.Designate one CSF sample as a “no primary” sample.

CRITICAL: This negative control sample is necessary for the analysis step of this protocol. The negative control sample(s) need to be processed to determine any signal resulting from any non-specific background. See Quantification and statistical analysis for more details.

• 61.Transfer 10 mL of each sample to a clean 1.5 mL Eppendorf tube.
• 62.Spin down CSF samples at 16,000 g with the Prism R Refrigerated Microcentrifuge (Labnet; Z723762).
• 63.Remove and dispose of the supernatant from each sample.

Optional: Since supernatant contains soluble proteins, rather than disposing, it can be used later to determine the levels of soluble proteins.

• 64.Add 250 μL of blocking solution to each tube (10% normal goat serum; MP Biomedicals, 191356) containing the pellet.

CRITICAL: The pellet may not be visible, so complete the blocking step.

• 65.Incubate the samples for 30 min with gentle rotation with the Roto-mini Rotator.
• 66.Spin down the samples at 16,000 g.
• 67.Remove and dispose of the resulting supernatant from each tube.
• 68.Add 50 μL of primary antibody to each sample (Adipogen, AG-25B-0006-C100).
• 69.Incubate the samples for 30 min with gentle rotation with the Roto-mini Rotator (Benchmark Scientific, R2020∗).

CRITICAL: Do NOT add any primary antibody to the “no primary” sample. Instead incubate it with Blocking Buffer.

CRITICAL: Do NOT remove primary antibody.

• 70.Add 250 μL of blocking solution to each sample to wash off residual unbound primary antibody.
• 71.Centrifuge the samples at 16,000 g.
• 72.Remove and dispose of the supernatant containing unbound primary antibody and blocking buffer.
• 73.Add 250 μL secondary antibody Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Thermo Fisher Scientific, A-21244) to each sample.
• 74.Incubate the samples for 30 min with gentle rotation using the Roto-mini Rotator.

CRITICAL: Do NOT remove secondary antibody.

• 75.Add 250 μL of blocking solution to each sample to wash off the primary antibody.
• 76.Centrifuge the samples at 16,000 g.
• 77.Remove the supernatant containing unbound secondary antibody and blocking buffer.
• 78.Resuspend the immunolabeled ASC speck pellet in 50 μL 1× PBS. Samples are now ready for flow cytometry.

Pause point

Note: While the sample may stay stable on ice for a few hours, acquiring images the day after staining has not been tested. It is recommended that staining and acquisition occur on the same day.

Visualization of ASC specks with an imaging-flow cytometer

Timing: 4 h

This section instructs users how visualize ASC specks with the flow-cytometer. It includes a description of how to turn on the flow-cytometer, select the laser settings, and navigate the software required to operate the ImageStream X Mark II.

Note: Information that includes screenshots and troubleshooting related to INSPIRE can be found in the INSPIRE ImageStreamX System Software User’s Manual.

• 79.Open the fluid bay to confirm that the storage containers have enough reagent for sample acquisition.
• 80.
  Turn on the imaging flow-cytometer.

  • a.
    Turn on the server connected to the flow-cytometer.
  • b.
    Wait a minimum of 2 min.
  • c.
    Press the green power button inside the front door of the ImageStream X Mark II to turn on the flow-cytometer.
  • d.
    Wait 2 min to proceed.
  • e.
    Turn on the computer.
  • f.
    Login with proper credentials provided by the vendor of the imaging flow-cytometer upon setup.
• 81.
  Launch INSPIRE software version 200.1.765.0 from the desktop. See troubleshooting 4.

  CRITICAL: Keep the script window open for INSPIRE software to continue running. This allows the flow-cytometer to communicate with the server.

  • a.
    Select “sequence startup” to initialize fluidics.

    Note: This takes approximately 20 min.

  • b.
    Allow the ASSIST calibration test to automatically begin.

    Note: This takes about 25 min.

  • c.
    Close the calibrations window once all the tests are passed.

    Note: As each calibration test passes, the status light for each test will change to green. See troubleshooting 5.

• 82.
  Confirm that the fluidics are running.

  • a.
    Display the “Fluidics” panel.
  • b.
    Set fluidics speed to “lo” to maximize the sensitivity of detection for best resolution (Figure S1).

CRITICAL: If samples are not ready, STOP the fluidics until samples can be acquired to conserve Speed Beads and reagents.

• 83.
  Load an “All Stain” sample.

  Note: Choose a sample that is the brightest biological condition among your samples. We recommend using stained purified ASC specks to optimize the laser settings.

  • a.
    Press “Load” to expose the sample holder.
  • b.
    Vortex sample briefly but thoroughly.
  • c.
    Place the 1.5 mL tube directly into the sample holder with the cap open and hinge seated in the hinge groove to assure proper fit and orientation of the tube.
  • d.
    Allow the following notification to pop-up, “Minimum volume to load 15 μL.”
  • e.
    Select “Okay”.

    Note: To reduce the risk of bubbles in the sample line, prepare at least 50 mL of volume per sample. Do not change this volume as it is the lowest volume the instrument will accept.

• 84.
  Define the target folder where your data will be stored (Figure S1).

  • a.
    Click the folder icon to the right of the file naming window in the “File Acquisition” panel.
  • b.
    Designate a name for your first file under the file name by typing it manually into the “filename” box.
• 85.
  Turn on the lasers needed for this protocol (Figure S1).

  • a.
    Display the “Illumination” panel.
  • b.
    Click the following lasers to turn them on:

    • i.
      405 nm for bright field (BF).

      Note: Ch1 & Ch9 are the default bright-field channel pairs, but if needed, other bright-field pairs can be chosen.

    • ii.
      642 nm for the ASC specks.
    • iii.
      785 nm laser for SSC.

      Note: It is recommended that SSC be assigned to Ch6. Ch12 can also be used for SSC.

• 86.
  Set the laser intensity for each laser (Figure S1).

  Note: To determine the optimal laser setting, stained purified ASC specks were used. In our hands, the following settings were optimized at 40× (Figure S1).

  • a.
    Type the following settings in manually:

    • i.
      405: 120 Mw.

      Note: By collecting bright field in one channel on each camera, images across each camera can be calibrated.

    • ii.
      642: 150 Mw.
    • iii.
      SSC: 1.95 Mw.

      Optional: Turn on additional lasers if other fluorescent fluorophores were added to the experiment.

  • b.
    Avoid any saturated pixels.

    • i.
      Use the image gallery to observe teal pseudo-colored areas (saturated pixels within objects).
    • ii.
      Lower the laser power of the appropriate channel if you see saturated pixels.

      Note: Refresh the image gallery to observe the impact of any new laser settings.

    • iii.
      Alternatively: Use the default scatter plots that display the Raw Max Pixel Intensity of each channel.

      Note: The histogram is displayed in the following pairs: Ch1 x Ch2, Ch3 x Ch4, Ch5 x Ch6, Ch7 x Ch8, Ch9 x Ch10, and Ch 11 x Ch12 (X and Y-axis, respectively).

    • iv.
      Reduce the laser power of the appropriate laser if objects on the scatter plot scatter collect at 4 x 10ˆ3 (saturated pixels).
• 87.
  Display the channels of interest.

  • a.
    Open the “Channel Selection Window.”
  • b.
    Select only the channels of interest.

    • i.
      Checkmark “1”, “6”, “9”, and “1”.

      CRITICAL: Only display necessary channels to reduce the file size of each sample.

• 88.
  Acquire Images (Figure S1). See troubleshooting 6, 7, 8, 9, and 10.

  • a.
    Manually set the “count” to “1000000” of “all” and collect “all”. See troubleshooting 11.

    CRITICAL: Do not gate during image acquisition. Collecting all objects ensures that data from every object is collected and not gated out until the analysis stage.

  • b.
    Select “Acquire” in the “Acquisition” panel.
  • c.
    Collect images for 4 min.

    Note: This should provide data on 5 μL of the sample. Since the flow rate is constant, this allows us to use the time acquired as a direct translation of the volume of sample.

    CRITICAL: Acquire images for the same amount of time for each sample to keep the volume collected uniform.

  • d.
    Select “Save File.”

Optional: Step 89 is optional.

• 89.
  Save Acquisition Settings as an .ist file.

  • a.
    Select “File”.
  • b.
    Click “Save as template.”
• 90.
  Return sample from machine.

  • a.
    Click “Return.”
  • b.
    Physically remove the 1.5 mL tube from the tube holder.
  • c.
    Select “Okay” to confirm to the software that the sample has been removed when prompted.

Note: The sample can be used again if necessary.

• 91.Repeat Steps 83–90 for remaining samples as necessary.

CRITICAL: Keep the acquisition settings uniform for each sample (ex. Laser power, magnification, flow speed, and acquisition time).

• 92.Confirm that data has been correctly stored and saved in the target folder as a raw image file (.rif).
• 93.Make a copy of the saved files so that they can be transferred to any unit that has IDEAS software.

Note: IDEAS 6.2 is not compatible with Mac OS systems.

Expected outcomes

Both ASC specks and Speed Beads should be observed while data collection is in progress (Figure 3). Purified ASC specks from ASC-cerulean macrophages cultured in the previous sections should have a high fluorescent signal. Conversely, a sample that is not stained with primary antibody should have a low fluorescent signal. Since the “no primary” control is also used for analysis, other negative controls can also be used for comparison. We recommend unstimulated ASC-cerulean macrophages or ASC knockout brain homogenate to be run in parallel with the CSF as additional controls.

Figure 3.

Representative image of ASC specks

(A) Example image of ASC specks when visualized (top) and Speed Beads (bottom).

(B) Size diagram of ASC specks and Speed Beads.

(C) Histogram that displays frequency of side-scatter (SSC) intensity when evaluating objects that are smaller than 5 μm and in focus. Two distinct populations are observed on the histogram when sorted by SSC intensity, the ASC specks, and the Speed Beads.

Here we report 1000% (10×) more ASC specks compared to a sample not stained with primary antibody. Once the analysis is conducted, the “no primary” sample should have a substantially lower number of objects identified as ASC specks. When at the data analysis stage, samples from patients with ADRD should have a significantly larger number of ASC specks in CSF (Figure 4A). To establish the importance of quantifying ASC specks in a sample, we demonstrate its usefulness in ADRD. ASC specks correlate with phosphorylated tau at threonine 181 (p-Tau181) and Aβ ratio (Aβ 42/40) in CSF (Figure 4B). The p-Tau 181 was measured with Fujirebio while Aβ ratio was determined with Meso Scale Discovery (MSD).⁴ Notably, this finding was corroborated with increased levels of soluble ASC (17 kDa) in ADRD CSF samples as determined by western blot analyses (Figure 4C).

Figure 4.

ASC specks can be quantified in human CSF

(A) When imaging flow cytometry is used to measure the level of ASC specks in human CSF samples, ASC specks are significantly elevated in patients with mixed-etiology dementia (n = 19) compared to controls (N = 11). Values are reported as mean ± SEM; Unpaired Student’s t-test; ∗p ≤ 0.05.

(B). ASC speck levels correlate with pathological hallmarks of dementia such as p-Tau 181; Pearson R = 0.4177; n = 9 controls and n = 28 dementia patients; ∗p ≤ 0.05. ASC specks also correlate with Aβ ratio (Aβ 42/40); Spearman R = -0.3877; n = 9 controls and n = 28 dementia; ∗p ≤ 0.05.

(C) Western blot corroborating that ASC specks are elevated in ADRD when 10 μL of sample is used. ASC knockout (ASC^−/−) brain homogenate is shown as a negative control.

Quantification and statistical analysis

Timing: 1 day

This section describes the gating method used to quantify the number of ASC specks in samples. Analysis includes the use of a gating strategy to determine the number of objects that meet the physical characteristics of ASC specks.

• 1.Launch IDEAS version 6.2 and proceed with gating one sample (Figure 5). For example, a positive control can be used.

Note: Purified ASC specks from ASC-cerulean macrophages cultured in the previous sections should have a high fluorescent signal. For this report, the example file has been named Sample 1.

• 2.
  Consider that the analysis makes use the following file types:

  • a.
    Raw image file (.rif): This file contains the high-resolution images taken during acquisition.
  • b.
    Compensated image file (.cif): This file applies a compensation matrix to the .rif data by calculating the number of pixels required to bring each channel into the perfect spatial registry.
  • c.
    Data analysis file (.daf): This file contains any histograms, scatterplots, gates, statistics, display, and report settings generated when analyzing the data.

Note: In order for IDEAS to use these files, the .rif, .cif, and .daf must have the same exact name and be in the same directory. Keep this in mind when conducting the analysis and saving files.

• 3.Open the .rif file.
• 4.Allow the compensated image file (.cif) to automatically be created.

Note: The software will automatically do this whether you have defined and applied a compensation matrix or not. It will also have the same name as the .rif file and be saved in the same directory as the .rif file, but with a .cif extension.

• 5.
  Use the gating strategy described in Figure 5 to generate a template analysis file (.ast) or .daf to later analyze merged .cif files.

  • a.
    Gate objects based on area (Figure 5A).

    • i.
      Select “new scatterplot” icon.
    • ii.
      Select title: primary sample 1.

      CRITICAL: The title of the graph defaults to the population represented on that graph.

    • iii.
      Select x-axis feature: Area_M01.
    • iv.
      Select y-axis feature: Aspect Ratio_M01.
    • v.
      Click “create rectangle region.”
    • vi.
      Select the region that is 1–20 μm² (the equivalent of 0–5 μm in diameter).

      Note: The scale of the graph will need to be adjusted. To do so, right click the graph. Select graph properties>scaling>manual. We recommend an X-axis minimum: −10 and maximum: 30.

    • vii.
      Manually name this region “S” for “small.”

      Note: All gates are manually named.

  • b.
    Select focused objects (Figure 5B).

    • i.
      Click the “new histogram” icon.
    • ii.
      Select x-axis feature: Gradient RMS_M01_BF.
    • iii.
      Click the “create line region” icon.
    • iv.
      Draw a linear gate over the area of the peak that includes most focused objects.

      Note: Exclude the most extremely unfocused objects at either end of the peak.

    • v.
      Name this region “focused small.”

      Note: To check what bins are in focus, use the image gallery. To view the selected bin in the image gallery, use the “Population” dropdown menu.

  • c.
    Gate out Speed Beads from all other objects (Figure 5C).

    • i.
      Click “new scatterplot.”
    • ii.
      Select x-axis feature: Intensity_M06 (which is SSC).
    • iii.
      Select y-axis feature: Intensity_M011 (which is channel 11 corresponding to an emission bandpass of 702/85 nm for Alexa 647).
    • iv.
      Create a rectangle region.
    • v.
      Select all objects that are SSC ^low to exclude the Speed Beads based on their SSC ^HI characteristic.
    • vi.
      Label the gate “not beads.”
• 6.
  Generate a histogram related to the fluorescence intensity of ASC specks (Figure 5D).

  • a.
    Click the “new histogram” icon.
  • b.
    Select the following x-axis-feature: Intensity_M011.

Pause point

Note: The file can now be saved as an .ast or .daf for determining the number of ASC specks.

• 7.
  Merge the “no primary” sample and its corresponding stained sample at the .cif level (Figure S2).

  • a.
    Select “tools>merge .cif files.”
  • b.
    Manually add the two correct .cif files.
  • c.
    Name the output files in a way that they can be easily identified and retrieved for analysis.

Note: For the purposes of this report, the example file names will be sample 1.cif and sample 2.cif (Figure S2). File names can be manually changed as needed.

• 8.Allow a merged data analysis file (.daf) to open with a blank layout to begin analysis.

Note: For the purpose of this example, the following files have been merged and a previously analyzed file is being used as a template (Figures S3–S7): Primary sample 1.cif and No primary sample 1.cif.

• 9.
  Use the gating hierarchy to correctly overlay histograms (Figures S3–S6).

  • a.
    Deselect the population automatically displayed.
  • b.
    Use “CTRL+click” to select the correct population.
  • c.
    Repeat the process of manually selecting the correct population for all gates (Supplementary 3–5).

CRITICAL: Due to merging files at the .cif level, the histogram will automatically set the maximum and minimum values of the x-axis, and therefore show the negative and positive peaks of both samples. This is why the populations must be manually selected.

• 10.
  Analyze the intensity of ASC Specks compared to background signal of the “no primary” sample.

  • a.
    Select “new histogram.”
  • b.
    Verify in the “Graph Properties” menu that the correctly displayed populations are selected in the gating hierarchy.

CRITICAL: Since this gate covers two distinct samples, the gating hierarchy should reflect a “not beads” population under the “No primary” parent population.

• 11.Right click on the histogram and choose “Populations.”

Note: Choose each population one at a time to change the color displayed on the graph. Right click “histogram smoothing” if desired.

• 12.
  Use histogram layering order to visually compare the Alexa 647 intensity of both the primary and “no primary” samples (Figure S6).

  • a.
    Right click the histogram.
  • b.
    Choose “Plot Order and Properties.”
  • c.
    Click the up or down arrow on the right side of the menu.

Note: This will move the selected population forward or backward in the layered histogram.

• 13.
  Change the line style of the histogram (Supplementary 6).

  • a.
    Right click on the graph.
  • b.
    Select “display properties.”
  • c.
    Uncheck the “Fill” box.
  • d.
    Select “dash dot” for line style.
• 14.
  Subtract any background signal using the “no primary sample.”

  • a.
    Verify that the signal of both samples is visible and distinguishable on the graph.
  • b.
    Use the line tool to select the region that is above the background of the “no primary” sample (Supplementary 7).

    CRITICAL: This is the region where the histograms of the two samples intersect.

  • c.
    Name this region “ASC++.”
  • d.
    Save this file as either a .daf OR .ast file.

    Pause point

    Note: This file can be used to subtract the value of the background signal, which is the number of ASC specks counted in the “no primary” sample.

• 15.
  Create a file to serve as a template for batch processing (Supplementary 8).

  • a.
    Open a new unmerged .rif file.
  • b.
    Follow the same gating paradigm established Steps 5 and 6.
  • c.
    Verify that an ASC specks fluorescent intensity histogram has been created (refer to Step 6).
  • d.
    Copy and paste the “ASC++” region of the merged .daf file to the template file.

    • i.
      Right click on the merged histogram.
    • ii.
      Select “Copy region to clipboard” (Figure S8).
    • iii.
      Right click on the unmerged .daf ASC intensity histogram.
    • iv.
      Select “Paste region to clipboard.”
    • v.
      Save the file as a .daf or .ast for later use as a template.

Note: The template can be used to batch process all .daf files.

Pause point

• 16.Batch process data files (Figure S9).
• 17.Graph data (See expected outcomes).

Figure 5.

Gating strategy required to quantify aggregated ASC specks

(A–D) The graph displayed includes one sample. (A) Objects are first gated based on size. This is done by creating a scatter plot that displays the area of the bright-field channel (Area_M01_BF) on the x-axis and aspect ratio of the bright-field channel on the y-axis (Aspect ratio_M01_BF). Objects that are 5 μm in diameter or smaller are selected and can be manually labeled “S” for small objects on the histogram. (B) The objects from the “S” population are further gated by creating a histogram of the gradient RMS of the bright-field channel (Gradient_RMS _M01_BF) to select objects that are in focus. (C) The objects that are small and in focus are further gated by generating a scatter plot that displays side scatter fluorescent intensity (SSC) on the x-axis and Alexa Flour 647 (ASC) intensity on the y-axis. This population can be manually labeled as, “not beads,” since they have a low side scatter. (D) Lastly, the SSC ^low objects are gated based on the ASC fluorescent intensity of the primary sample and no primary sample. Objects that are above background are determined by gating the area of the histogram where the no primary sample and primary sample intersect. This effectively subtracts the area highlighted in yellow.

Limitations

Analyzing CSF samples with imaging flow cytometry enables the quantification and analysis of CNS-derived ASC specks. However, obtaining consent for spinal taps from healthy control subjects and those in rural communities may be a major hurdle. Access to MSD and imaging flow-cytometers in research laboratories is uncommon in these communities as well. Future work will therefore incorporate the use of plasma and serum since they are obtained through less invasive means. This protocol has only been optimized for CSF and is not recommended for other biological samples such as plasma, serum, or brain homogenates. Another limitation of the protocol is the concentration of the primary antibody. Note that using the antibody at a higher concentration may lead to non-specific binding in the samples. Therefore, careful titration of the antibodies is necessary prior to adapting this protocol for clinical diagnosis purposes.

There are also limitations to this study related to the imaging flow-cytometer. For example, the imaging flow-cytometer used to establish this protocol does not have high gain functionality or 60× magnification. Lastly, substantial storage space is necessary to store the large files obtained during image acquisition. To prevent the computer from crashing during the run or analyses, the imaging flow-cytometer must be connected to a large server or external hard drive.

Overall, it is essential to consider how these limitations may be overcome by adapting the protocol. For example, the gating parameters may be extended to other flow cytometers, Speed Beads, or biological samples. Antibody concentrations may also be tested at different concentrations (See Alternatives).

Troubleshooting

Problem 1

ASC-cerulean cells do not adhere to the T-75 flask well after thawing a fresh vial out or splitting the cells.

Potential solution

Make fresh media in case all the necessary growth factors were not previously added. Pre-coat the flask or cell culture dish with adherence factors such as laminin or poly-D-lysine. Another brand of cell culture dish can be purchased as well.

Problem 2

ASC-cerulean cells are no longer adherent to the cell culture dish after activating the inflammasome with nigericin stimulation.

Potential solution

Harvest the media that contains the cells that are in suspension and spin down the cells as recommended in this protocol. If the problem persists, consider pre-coating the flasks as suggested in problem 1.

Problem 3

The concentration of ASC specks isolated from ASC-cerulean macrophages is low.

Potential solution

Expand the number of flasks and cell culture dishes so that more cells are placed through the isolation method. If this does not work, then consider obtaining ASC specks through another purification method. For example, an immunoprecipitation-based technique.²

Note: The list below was adapted from the INSPIRE ImageStreamX System Software User’s Manual. For additional instructions or troubleshooting please refer to the same manual.

Problem 4

INSPIRE application does not open.

Potential solution

Use Windows Task Manager to end INSPIRE. If this does not work, INSPIRE may not be communicating correctly with the imaging flow-cytometer. To reconnect the equipment, restart the entire system including the computer and flow-cytometer. Wait 5 min after the shutdown to restart the equipment.

Problem 5

INSPIRE flags the failure of one or more calibration tests.

Potential solution

Determine which tests have failed before proceeding. First verify that the flow-cytometer is on by checking if the power button on the flow-cytometer is green. If the machine is on, make sure that the tubing is not clogged. To unclog the tubing, run the “Purge Bubbles” script. Once it is verified that the tubing is cleared, confirm that the Speed Beads are running. If the Speed Beads are not running, return any sample and check the fluidics. Change the flow speed first, and subsequently stop and restart the fluidics since the beads may not be well mixed. If the problem persists, replace the Speed Beads in the fluid bay. “Spatial offset” or “focus adjuster” tests may also fail. If those fail, verify that bright field is functioning correctly. Lastly, the “dark current calibration” test may fail. If this occurs, verify that none of the lasers are turned on. Turn off the flow-cytometer and restart the system if this is the case. If any of the tests for a specific laser fails, confirm that the laser can turn on.

Problem 6

ASC Specks are not observed in the CSF sample (See expected outcomes).

Potential solution

Verify that the correct lasers are turned on. If they are on, maximize the laser power until ASC specks are visible in a positive control sample. Subsequently lower the laser power if the pixels are saturated. If either Speed Beads or ASC specks are not observed, the position of the core stream may be a problem. It can be manually corrected by manipulating the arrows available on the “Focus and Centering” panel. Confirm that the frame is in focus as well. If the problem continues, it is likely that primary antibody or secondary antibody was not added to the sample. Try running another sample with the flow-cytometer to verify if other samples are visible on the screen.

Problem 7

Objects are not in focus or blurry.

Potential solution

First allow the system to settle for 60 s after loading a sample. If the problem persists after the system has settled, re-run tests on ASSIST such as the “focus adjuster” and “frame offset” calibration tests. Confirm that the tests are passed. If the tests are passed but the issue continues, check the “Focus and Centering” panel to adjust focus and centering as described in Problem 9. Also verify that the system is not clogged. To that end, use the instrument drop-down menu to run the “Purge Bubbles” script. Make sure to also purge any air bubbles that may be causing an obstruction. If the system is not clogged, stop acquisition, and replace the 1× PBS sheath fluid. It may be contaminated.

Problem 8

The Speed Beads are not flowing in a uniform manner and appear to be clumped or sporadic. The fluidics are not stable.

Potential solution

Make sure that the conical of Speed Beads is not empty, and immediately replace it if necessary. If beads are present, the vortex that mixes the Speed Beads during data collection may be broken. Gently mix the beads between samples if image acquisition is not complete. Once the experiment is done, immediately contact the appropriate vendor for repair. The tubing may also be clogged. To clear the air bubble, run the “Purge Bubbles” script.

CRITICAL: If image acquisition is interrupted in scenarios like this, it is important to repeat the experiment to confirm that the data is reproducible.

Problem 9

Objects are out of focus.

Either the ASC specks or Speed Beads appear to be offset too far to the left or the right of the frame. Alternatively, neither may appear in the frame.

Potential solution

First allow the system to settle for 60 s after loading a sample to ensure that autofocus can occur. The camera line rate may be incorrect. Re-run the “focus adjuster” and “frame offset calibration” tests in ASSIST. Alternatively, verify that the system is not clogged. To clear any air bubbles, run the “Purge Bubbles” script.

Problem 10

Objects in the frame resemble streaks.

Potential solution

Check that bright field is captured on both cameras. To do this, confirm that 1 and 9 are selected as “Brightfield” in the “Illumination” panel (Figure S2). In case the Speed Beads have stopped running, double-check their status by experimenting with the fluidics. For example, return any sample that is running and stop the fluidics in the “Fluidics” panel. Restart the fluidics after a 10 s pause and select the flow speed.

Problem 11

Slow and erratic imaging is causing INSPIRE to crash.

Potential solution

Check that the camera is turned on. If it is off, click “Run>Setup” to turn it on. If the camera is on, click “stop” before selecting “Run>Setup.” Confirm that data collection was not incorrectly paused. If so, press “resume” to continue image acquisition. If INSPIRE has crashed, use Windows Task Manager to end INSPIRE. Once the program is completely shut down, restart the program, and verify that all lasers and the bright-field lamp are turned on.

CRITICAL: If INSPIRE has crashed, check the .rif file for errors. For example, the object number may be displayed as zero in the .daf file. To fix the .rif file, use the “Merge .rif file” command. To that end select “Tools>Merge .rif Files.” Click “Add Files” to select the .rif file that is damaged. Click “OK”. This will rewrite the .rif file with the correct object count.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Kiran Bhaskar (kbhaskar@salud.unm.edu).

Technical contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the technical contact, Kathryn Sánchez (Kathryn.Sanchez@bsd.uchicago.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

Original/source data for [figures/datatype] in the paper is available on Mendeley Data [Sanchez, Kathryn (2023), “Cell STAR Methods”, Mendeley Data, V1, https://doi.org/10.17632/btg6tcryt8.1].