Retro-orbital CD45 antibody labeling to evaluate antibody-secreting cell trafficking in mice

Summary

Antibody-secreting cells (ASCs) are critical regulators of the humoral immune response. However, differences between tissue resident populations versus those that have recently migrated to their final anatomic destination are poorly understood. Here, we present a protocol for using retro-orbital (r.o.) CD45 antibody labeling to identify tissue resident versus recently immigrated ASCs in mice. We describe steps for r.o. injection of antibodies, animal euthanasia, and tissue harvesting. We then detail tissue processing, cell counting, and cell staining for flow cytometry analysis.

For complete details on the use and execution of this protocol, please refer to Pioli et al. (2023).¹

Graphical abstract

Highlights

• •Protocol for r.o. CD45-PE antibody labeling to measure ASC trafficking
• •Steps to harvest and process tissues for cell staining
• •Steps for surface staining of cells preceding flow cytometric analysis
• •Examples of how to analyze and interpret flow cytometry data

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

Antibody-secreting cells (ASCs) are critical regulators of the humoral immune response. However, differences between tissue resident populations versus those that have recently migrated to their final anatomic destination are poorly understood. Here, we present a protocol for using retro-orbital (r.o.) CD45 antibody labeling to identify tissue resident versus recently immigrated ASCs in mice. We describe steps for r.o. injection of antibodies, animal euthanasia, and tissue harvesting. We then detail tissue processing, cell counting, and cell staining for flow cytometry analysis.

Before you begin

The protocol described below focuses on using 5 min r.o. CD45-phycoerythrin (PE) antibody labeling to examine ASC trafficking. The inclusion of alternative lineage-specific markers will adapt this basic protocol to your cell type of interest, assuming the cells express CD45. While this protocol was originally developed (and is detailed below) using 3 months old female and male Prdm1-eYFP reporter mice,² other strains of mice (e.g., wild-type C57BL/6J) can be utilized. Finally, this protocol discusses the use of a 5 min labeling period. However, this timing can be altered to suit the experimental question being asked.

Institutional permissions

All animal experiments require the approval of your institutional animal care and use committee. As such, none of the experiments described here can be performed until that approval is in place.

Reserve anesthesia machine for use in your vivarium

Timing: 5 min

• 1.Verify access to anesthesia equipment for the day and time that you plan to perform the experiment.

CRITICAL: All injections will be performed via the r.o. route necessitating that animals are under proper anesthesia. Do not perform this protocol until you are trained to properly operate anesthesia equipment and perform r.o. injections. These points are critical to preserving the overall health and safety of the animals used for experimentation.

Prepare CD45-PE antibody solution

Timing: 15–30 min

• 2.
  Using the C₁V₁ = C₂V₂ formula, determine CD45-PE antibody dilution to deliver 1 μg of antibody per mouse in a 100 μL volume (final antibody concentration = 10 μg/mL). Calculations are made incorporating an additional replicate (n + 1) for precautionary reasons.

  • a.
    Example: injecting 3 mice using an antibody with the stock concentration of 0.2 mg/mL (or 200 μg/mL).

    • i.
      Definitions for C₁V₁ = C₂V₂ formula:
       C₁ = concentration of stock antibody.
       V₁ = volume of stock antibody.
       C₂ = desired/final concentration of antibody.
       V₂ = desired/final volume of antibody.
    • ii.
      (200 μg/mL)(V₁) = (10 μg/mL)(0.4 mL).
       V₁ = (10 μg/mL ∗ 0.4 mL) / (200 μg/mL) = 0.02 mL = 20 μL stock antibody.

• 3.
  Dilute stock antibody using sterile 1× PBS as calculated above.

  • a.
    Set aside an additional aliquot of sterile 1× PBS as this will be injected into mice as a negative control for CD45-PE (r.o.) staining.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
CD45-PE (Clone: 30-F11) (1 μg injected per mouse, working concentration of 10 μg/mL)	BioLegend	Cat# 103106; RRID: AB_312971
CD45-APC (Clone: 30-F11) (0.05 μg used per stained sample)	BioLegend	Cat# 103112; RRID: AB_312977
CD138-BV421 (Clone: 281-2) (0.02 μg used per stained sample)	BD Biosciences	Cat# 562610; RRID: AB_11153126
IgD-BV605 (Clone: 11-26c.2a) (0.04 μg used per stained sample)	BioLegend	Cat# 405727; RRID: AB_2562887
CD90.2-BV605 (Clone: 53-2.1) (0.04 μg used per stained sample)	BD Biosciences	Cat# 563008; RRID: AB_2665477
CD45R(B220)-PerCP/Cy5.5 (Clone: RA3-6B2) (0.20 μg used per stained sample)	BD Biosciences	Cat# 552771; RRID: AB_394457
CD19-BUV395 (Clone: 1D3) (0.60 μg used per stained sample)	BD Biosciences	Cat# 563557; RRID: AB_2722495
CD16/32-Unlabeled (Clone: 93) (Fc receptor blocking) (1.00 μg used per stained sample)	Thermo Fisher Scientific	Cat# 14-0161-86; RRID: AB_467135
Chemicals, peptides, and recombinant proteins
Bovine serum albumin (DNase- and protease-free)	Fisher Scientific	Cat# BP9706100
Dulbecco’s phosphate buffered saline powder (calcium and magnesium free)	Gibco	Cat# 21600-010
AErrane (isoflurane) 100% liquid	Baxter	Cat# CA2L9108
NH4Cl	Fisher Scientific	Cat# A661-500
KHCO3	Fisher Scientific	Cat# P235-500
Na2-EDTA	Fisher Scientific	Cat# BP120-1
Trypan Blue powder	Fisher Scientific	Cat# AC189350250
Experimental models: Organisms/strains
Mouse: B6.Cg-Tg(Prdm1-EYFP)1Mnz/J (female and male mice heterozygous for the Prdm1-eYFP transgene were used at 3 months of age)	The Jackson Laboratory	JAX: 008828; RRID: IMSR_JAX:008828
Software and algorithms
FlowJo (v10)	BD Biosciences	RRID: SCR_008520
Other
Frosted microscope slides	Fisher Scientific	Cat# 22-034-486
BD lo-dose U-100 insulin syringes w/ 28G needle	Fisher Scientific	Cat# 14-826-79
60-mm sterile petri dishes	Fisher Scientific	Cat# FB0875713A
BD vacutainer collection tubes	Fisher Scientific	Cat# 02-683-45
5-mL polystyrene test tubes	Fisher Scientific	Cat# FB1496110
5-mL polystyrene test tubes with cell strainer snap cap	Fisher Scientific	Cat# 08-771-23
5-mL BD syringes with Luer-Lok	Fisher Scientific	Cat# 14-829-45
BD hypodermic needles (23G)	Fisher Scientific	Cat# 14-826-6B
40-μm sterile cell strainers	Fisher Scientific	Cat# 22-363-547
Veterinary anesthesia machine	Dispomed	https://www.dispomed.com/product-category/veterinary-anesthesia/veterinary-anesthesia-machines/
BD LSRFortessa flow cytometer	BD Biosciences	https://www.bdbiosciences.com/en-ca/products/instruments/flow-cytometers/research-cell-analyzers/bd-lsrfortessa
Sorvall X4R Pro-MD refrigerated benchtop centrifuge	Thermo Fisher Scientific	Cat# 75009521

Materials and equipment

Alternatives: This protocol uses a 5-laser BD LSRFortessa to analyze samples stained with the fluorochrome-conjugated antibodies detailed above. However, fluorochromes are amenable based upon the spectral capabilities of the available flow cytometer.

Red Blood Cell (RBC) Lysis Buffer (a.k.a., ACK Lysis Buffer)

Reagent	Final concentration	Amount
NH4Cl	150 mM	8.02 g
KHCO3	10 mM	1.001 g
Na2-EDTA	0.1 mM	37.22 mg
ddH2O	N/A	1000 mL
Total	N/A	1000 mL

Note: pH to 7.2–7.4. Store at 4°C for up to 6 months. Solution can be filter sterilized if working with sterile tissues/cells.

Flow Staining Buffer (FSB)

Reagent	Final concentration	Amount
BSA	0.1%	1 g
1x PBS	N/A	1000 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to 6 months. Solution can be filter sterilized if working with sterile tissues/cells.

Flow Running Buffer (FRB)

Reagent	Final concentration	Amount
BSA	0.1%	1 g
Na2-EDTA	2 mM	744.48 mg
1× PBS	N/A	1000 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to 6 months. Solution can be filter sterilized if working with sterile tissues/cells.

Alternatives: Fetal bovine serum (FBS) can be substituted for BSA in both the Flow Staining Buffer and Flow Running Buffer.

• •0.4% (w/v) Trypan Blue: mix 0.4 g Trypan Blue into 100 mL 1× PBS.

Note: Filter sterilize solution and store at 20°C–25°C. Solution can be stored for up to 1 year.

Step-by-step method details

Retro-orbital injection of CD45-PE antibodies, animal euthanasia and tissue harvesting

Timing: 15–30 min per mouse

This section details steps progressing from r.o. injection of CD45-PE antibodies (or just 1× PBS) to blood and tissue collection.

• 1.
  Anesthetize mouse using a veterinary anesthesia machine (Figure 1A).

  • a.
    Set oxygen at maximal input.
  • b.
    Place mouse in anesthesia chamber (Figure 1B).
  • c.
    Infuse anesthesia chamber with isoflurane (5%).
  • d.
    Allow mouse to obtain a slow and steady breathing pattern (Methods video S1).
• 2.Remove animal from chamber and place on its side (Figure 1C).
• 3.Using an insulin syringe with 28G needle, inject 100 μL of 1× PBS or CD45-PE antibodies (10 μg/mL) (Figures 1D–1F).

Note: Recommended maximum volume per mouse is 150 μL and 10 μL for adults and neonates,³ respectively. Please note that lower volumes such as 50 μL can be utilized reliably in adult mice.

• 4.Place animal into separate cage and start timer (previously set to 5 min).
• 5.At time, euthanize mouse via CO₂ inhalation (primary form of euthanasia).
• 6.At cessation of breathing (∼30–60 s), remove animal from chamber and apply footpad pressure to verify non-responsiveness.
• 7.Using a dissection board (e.g., Styrofoam covered with tin foil and/or paper towels), position mouse with ventral side facing up and limbs spread (Figure 1G).
• 8.Spray mouse with 70% ethanol (EtOH).
• 9.Using scissors and forceps, expose internal organs (Figure 1H) and perform cardiac puncture (Figure 1I) as secondary form of euthanasia.

Note: To expose the heart, cut horizontally along bottom of ribs and diaphragm. Starting from the bottom of the rib cage, make 2 vertical cuts along the lateral portions, “flip up” the center portion of the rib cage and pin down to stabilize (Figure 1I). Cardiac puncture is performed by inserting a needle into the left ventricle and blood is harvested by slowly drawing back on the syringe plunger. To reduce the initial pressure vacuum, it is recommended to cycle the syringe plunger back and forth before ventricular insertion. This is especially important when using small bore needles such as a 28G insulin syringe.

Alternatives: If cardiac puncture is not feasible, cervical dislocation can be performed as a means of secondary euthanasia. The mouse is then dissected, and blood collected following aortic bisection.

• 10.Transfer blood to BD Vacutainer tube (or equivalent blood collection tube), invert multiple times to mix and place on ice.
• 11.Dissect organs of choice and place them on ice into individual 60-mm petri dishes filled with 5 mL of 1× PBS.

CRITICAL: Each experiment requires at least 1 animal that receives only 1× PBS. Samples derived from these mice will be essential in establishing background fluorescence in the PE channel upon flow cytometric analysis.

CRITICAL: Given the processing time for 1 mouse, it may be preferential to work in teams and assign 1 task per individual. If working with more than 2 mice, this will allow the team to stagger animals and perform the labeling and tissue harvesting in a more efficient manner.

Figure 1.

Representative images depicting animal anesthesia, r.o. injection, and cardiac puncture

(A) Representative image of anesthesia machine.

(B) Image showing mouse inside isoflurane anesthesia chamber. Bottom of chamber is lined with paper towels to collect any excrement.

(C) Pre-injection: place mouse on its side with the r.o. cavity positioned upward.

(D) Using thumb and index finger, stabilize the skull and apply gentle pressure on the fur/skin proximally located above and beneath the eye. This increases the perceived space between the eye and r.o. cavity allowing for easier injection.

(E) Slowly insert the needle into the r.o. cavity until a slight bump is felt. This indicates the region where ophthalmic veins join. Depress plunger and deliver payload while applying slight downward pressure to penetrate/puncture the veins.

(F) Upon delivery, transient eye “bulging” will be observed due to the increased liquid volume behind the eye. However, no leakage should be observed if injection was successful.

(G) Image showing mouse position for dissection.

(H) Exposure of mouse internal organs.

(I) Image showing cardiac puncture with rib cage “flipped up” and stabilized using dissection pin.

Tissue processing

Timing: 1–2 h (dependent on number of animals)

This section details the processing of tissues post-harvesting.

• 12.
  Organ processing.

  • a.
    Solid organ (e.g., spleen (SPL), thymus (THY)): In 60-mm petri dish with 5 mL 1× PBS, crush organ in between frosted ends of 2 microscope slides and transfer cell suspension to 15-mL conical tube on ice.
  • b.
    Bone marrow (BM): In 60-mm petri dish with 5 mL 1× PBS, cut off ends of bones with dissection scissors (or razor blade) and flush BM contents using 23G needle mounted onto a 5-mL syringe. Transfer cell suspension to 15-mL conical tube on ice.

Note: We typically flush both femurs and tibias which provides more than adequate numbers of cells required for the analysis described below.

• 13.Centrifuge samples at 600 g for 5 min at 4°C.
• 14.Decant supernatant and resuspend cells in residual buffer (∼100 μL) by raking tubes against the rungs of a metal tube rack.

Alternatives: Cells can also be resuspended by finger flicking of tubes combined with gentle pipetting.

• 15.
  Add 3 mL of RBC Lysis Buffer, mix thoroughly (e.g., pipetting or tube inversion) and place samples on ice for 3 min.

  • a.
    For blood: 3 mL RBC Lysis Buffer is directly added to 100 μL of blood in 15-mL conical.

Note: RBC lysis buffer is stored at 4°C for up to 6 months.

Note: RBC lysis of peripheral blood within this protocol is usually incomplete. However, it reduces RBC density sufficiently to allow for flow cytometry. It also does not result in the potential loss of leukocytes which may occur when performing gradient-based separation (e.g., Percoll). If complete RBC lysis is desired, incubation times can be extended. However, this should be tested beforehand to verify that other cells are not inadvertently lysed as a result of increased time in the RBC Lysis Buffer.

• 16.Add 7 mL of 1× PBS to neutralize RBC lysis buffer.
• 17.Pass cells through a 40-μm cell strainer into new conical tube on ice.

• 18.
  Count cells using Trypan Blue and a Hemocytometer.

  • a.
    In general, blood is omitted from this count as mouse blood normally contains from 2,000–10,000 leukocytes/μL.⁴ Based upon this, the cellular concentration is too low to count using this method. If mouse blood cell counts are needed, samples can be run on complete blood cell (CBC) machine.
  • b.
    Follow the below steps to counts cells:

    • i.
      Dilute samples 1:1 in Trypan Blue (this represents a 2-fold dilution).

      Note: If sample is too concentrated to count, dilute before adding Trypan Blue. Make a notation of this number as it will contribute to the final dilution factor used below to calculate cells/mL.

      Note: Trypan Blue is stored at 20°C–25°C for up to 1 year.

    • ii.
      Load Hemocytometer with 10 μL of cells plus Trypan Blue mixture (Figure 2).
    • iii.
      Using a 20× microscope objective, count “clear” cells in 4 grids (Figure 2). “Clear” cells represent live cells while “blue” cells have taken up Trypan Blue dye as a result of a loss of membrane integrity. As such, “blue” cells are considered apoptotic.
    • iv.
      Calculate cells/mL using the following formula: (total live cells counted / number of grids) × dilution factor × 10,000 = cells/mL
    • v.
      Calculate the total cells per organ/sample using the following formula: cells/mL × total sample volume = total cells per organ/sample

      CRITICAL: The total cells per organ/sample will be used for 2 downstream calculations. (1) Determination of appropriate resuspension volume of samples before flow staining (relevant to step-by-step method details step 20). (2) Calculating absolute numbers of populations of interest following flow cytometry (relevant to quantification and statistical analysis).

      Alternatives: Cells can also be counted using automated systems such as a Countess.

• 19.Centrifuge samples at 600 g for 5 min at 4°C.
• 20.
  Decant supernatant and resuspend in Flow Staining Buffer (FSB, 1× PBS + 0.1% BSA) at a concentration of 2 × 10⁷ cells/mL.

  • a.
    Due to lower relative cellularity, blood samples can be resuspended in a minimal volume (e.g., 250 μL).

Note: FSB is stored at 4°C for up to 6 months.

CRITICAL: Perform all steps in a biosafety cabinet using pre-sterilized solutions if cells will be subsequently cultured. Avoid direct light when processing samples to prevent potential quenching of PE fluorescence.

Pause point: Cells can be kept on ice for an extended period to allow for a break and/or preparation of downstream reagents.

Figure 2.

Representative counting of cells using a Hemocytometer

Schematic shows loading of a Hemocytometer with a 1:1 mixture of cells:Trypan Blue (2-fold dilution). Live cells are “clear” and apoptotic cells are “blue”. Cells are counted in the upper left, upper right, lower left and lower right grids. If cells sit on edges of a grid, then only 2 out of 4 edges are considered as part of the grid. This is done to prevent over estimation of cell counts. In this schematic, cells on the top and right edges are considered as part of the grid. Cells/mL is calculated as follows: (total live cells counted / number of grids) × dilution factor × 10,000 = cells/mL. Using this example, cells/mL = (10 / 4) × 2 × 10,000 = 50,000. Figure made with BioRender.

Cell staining for flow cytometry

Timing: 1–2 h (dependent on number of samples)

This section details the surface staining of cells for flow cytometric staining. However, these steps can be adapted to include intracellular staining when necessary (adds ∼1–2 h). For intracellular staining, it is recommended that antibodies and fluorochromes used for surface staining are tested for their resistance to fixatives as well as cell permeabilization reagents.

• 21.
  Prepare Antibody Cocktail used for staining.

  • a.
    All antibodies are diluted in 1× PBS.

    Note: All antibody stock tubes are briefly vortexed, and touch spun before use to properly mix. All antibody stocks are opened only in a biosafety cabinet in the dark to preserve their sterility and prevent quenching of fluorescence.

  • b.
    For example, Antibody Cocktail is shown to stain 1 sample. However, it is normally scaled up to stain n + 1 samples.
    Antibody Cocktail

    Antibody	Stock concentration (μg/μL)	Dilution from stock	Volume used per sample	Amount of antibody used (μg)
    CD45-APC	0.2	1:40	10 μL	0.05
    CD138-BV421	0.2	1:100	10 μL	0.02
    IgD-BV605	0.2	1:50	10 μL	0.04
    CD90.2-BV605	0.2	1:50	10 μL	0.04
    CD45R(B220)-PerCP/Cy5.5	0.2	1:10	10 μL	0.20
    CD19-BUV395	0.2	No Dilution	3 μL	0.60
    CD16/32-Unlabeled (Fc receptor blocking)	0.5	No Dilution	2 μL	1.00
    Total Volume per Sample		N/A	55 μL	N/A

    Note: If no dilution, the Volume Used per Sample corresponds to the volume taken straight from the antibody stock tube.

    Note: Amount of Antibody Used (μg) is titrated for staining samples up to 5 × 10⁶ cells. These amounts can be scaled up for staining of increased cell numbers (e.g., scale each antibody by a factor of 2 if staining 1 × 10⁷ cells). Additionally, these amounts may vary depending on flow cytometer and laser setup/parameters. The Volume Used per Sample for each antibody is independent of the total cocktail volume. For example, addition of another antibody (e.g., 10 μL of diluted CD23) does not change the amount/volume of other antibodies within the total Antibody Cocktail.

    Note:Ex vivo CD45-APC staining is included to confirm CD45 surface expression on cell types of interest. Additional surface markers can be included to probe cellular phenotypes such as activation.

  • c.
    Vortex Antibody Cocktail to mix, briefly touch spin and hold on ice (in the dark) until use).

• 22.
  Aliquot 250 μL (5 × 10⁶ cells) per sample to be stained into a 5-mL test tube on ice.

  • a.
    For blood, 250 μL will correspond to < 5 × 10⁶ cells.
• 23.Add 55 μL of Antibody Cocktail to each a sample.

Note: Fc receptor blocking is performed via the CD16/32-Unlabeled antibody included within the Antibody Cocktail.

• 24.Touch vortex and incubate on ice (in the dark) for 30 min.
• 25.Add 3 mL FSB per sample and centrifuge at 600 g for 5 min at 4°C.
• 26.Decant supernatant and resuspend in residual buffer (∼100 μL).
• 27.Add 250 μL of Flow Running Buffer (FRB, 1× PBS + 0.1% BSA + 2 mM EDTA).

Note: The inclusion of EDTA in the FRB helps to reduce cell clumping. Volume of FRB is variable depending on the desired final cell density per sample.

Note: FRB is stored at 4°C for up to 6 months.

• 28.Strain samples into new 5-mL test tube via attached snap cap strainer.

Alternatives: Generic nylon mesh or other single use strainers can be substituted.

CRITICAL: Cell straining is a key step to eliminate clumps that may have formed during sample processing. This helps to reduce the potential of clogging the flow cytometer during use.

• 29.Run samples on flow cytometer and collect data for downstream analysis using FlowJo.

Note: When running flow cytometry, setting the “stopping gate” based upon ASCs rather than total events will allow the user to ensure that enough ASCs are collected for reliable downstream analysis.

Expected outcomes

The results presented here are based upon data that were originally acquired in Pioli et al., 2023.¹ It is expected that approximately 99% of peripheral blood cells will stain CD45-PE (r.o.)⁺ after the 5 min labeling period. This is an important observation and validates the success of the r.o. labeling protocol. Labeling is expected to vary depending on the type of cell and organ being analyzed. For example, cells that routinely circulate between organs will demonstrate significant CD45-PE (r.o.) labeling. In contrast, cells with a tissue resident phenotype are non-circulatory and will remain unlabeled.

Quantification and statistical analysis

Analysis is performed on live single cells which are gated as demonstrated in Figure 3A. In this instance, live cells are identified solely based upon forward and side scatter characteristics. Live-Dead staining reagents can be included as an additional method to gate viable cells. Analysis of only live cells is critical as dead cells have increased autofluorescence and tend to be “sticky” regarding non-specific antibody binding. As such, inclusion of dead cells can skew your results. The next step is to verify the success of r.o. labeling by assessing the level of CD45-PE antibody staining in the blood. Samples from PBS treated mice serve as a measure of background fluorescence (Figure 3B). Figure 3B shows multiple ways in which samples from PBS and CD45-PE antibody treated mice can be visualized and gated. The left panel in Figure 3B compares CD45-APC (ex vivo) versus CD45-PE (r.o.) staining. Examination of the data from this perspective confirms CD45 surface expression on the sample being analyzed. The right panel in Figure 3B shows CD45-PE (r.o.) staining using a histogram to demonstrate the overall shift in PE geometric mean fluorescence intensity (gMFI). This gMFI shift is readily apparent in peripheral blood cells isolated from mice that received CD45-PE antibodies. Both visualization methods confirm the efficacy of the r.o. labeling protocol.

Figure 3.

Representative singlet gating and validation of CD45-PE (r.o.) labeling in the blood

(A) Representative gating of singlets using peripheral blood as an example.

(B) Validation of CD45-PE (r.o.) labeling in peripheral blood. Left and right panels overlay blood samples from PBS and CD45-PE antibody treated mice. Left panel shows CD45-APC (ex vivo) versus CD45-PE (r.o.) staining. Right panel shows CD45-PE (r.o.) staining as a histogram. Percentages shown indicate CD45-PE (r.o.)⁺ cells from blood of CD45-PE antibody treated mice.

Next, ASCs are gated in the various samples being assayed (Figure 4). Like Figure 3, CD45-PE (r.o.) staining can be assessed using 2-parameter plots (e.g., dot, contour) or histograms. The use of histograms is shown in Figure 5A to demonstrate the feasibility to make CD45-PE (r.o.) labeling comparisons amongst ASCs from different organs. In this context, positive staining cells are obvious in at least 1 of the samples. However, if CD45-PE (r.o.) labeling is limited in every population, then 2-parameter plots provide better visualization of rare events. Figure 5B shows expression of CD45-APC (ex vivo) and CD45-PE (r.o.) for THY immature B cells (CD45R(B220)^INT CD19^INT CD138^-), mature B cells (CD45R(B220)^HI CD19^HI CD138^-) and ASCs. All 3 populations from PBS treated and CD45-PE antibody treated mice are shown as overlays (Figure 5B). Using this approach, rare events are readily apparent to the eye.

Figure 4.

Representative gating of ASCs in the BM, SPL, and THY

Gating is shown in the context of Prdm1-eYFP reporter mice and ASCs are identified as CD138^HI IgD^-/LO CD90.2^-/LO Prdm1-eYFP⁺. IgD expression has been observed for ASCs localized in mucosal tissues. As such, this parameter needs to be tested before inclusion in the Antibody Cocktail. If working with non-Prdm1-eYFP mice, cell surface markers such as CD267(TACI) and CD44 can be used to gate ASCs. All samples are pre-gated on live singlets and numbers in plots represent percentages within parent populations.

Figure 5.

Representative plots depicting methods to compare ASC populations or ASCs versus other cell types

(A) Histogram overlaying CD45-PE (r.o.) staining of ASCs from BM, SPL and THY. Samples from PBS and CD45-PE antibody treated mice are shown. Red line added to histogram shows cut-off for positive staining. Numbers indicate % of CD45-PE (r.o.)⁺ cells within each sample.

(B) Dot plots showing CD45-APC (ex vivo) versus CD45-PE (r.o.) staining. PBS and CD45-PE antibody treated samples are overlayed for THY immature B cells, mature B cells, and ASCs. Numbers indicate % of CD45-PE (r.o.)⁺ cells within each sample.

Finally, quantification is straight forward and requires a background (i.e., PBS sample) subtraction calculation as shown in Table 1. This results in the % of CD45-PE (r.o.)⁺ cells per population of interest. To calculate the total number of labeled cells per population, multiply the background subtracted percentage labeled by the number of cells per population of interest (Table 1, e.g., THY Mature B: (0.55% ∗ 100,000 = 550 CD45-PE (r.o.)⁺ THY Mature B cells). The number of Cells per Population is calculated as (% of population within total live single cells (derived from FlowJo) ∗ total cells per organ/sample) (step not shown). Please note that the total cells per organ/sample is calculated in step-by-step method details step 18.

Table 1.

Example of CD45-PE (r.o.) calculations using hypothetical values

Sample	% CD45-PE (r.o.)+			Cells per population	Number of CD45-PE (r.o.)+ cells
	CD45-PE treated	PBS treated	Background subtracted
THY Mature B	0.65	0.10	0.55	100,000	550
THY ASC	0.0	0.0	0.0	10,000	0
SPL ASC	44.5	1.5	43.0	250,000	107,500

Limitations

The major limitation of this protocol is that it relies on the expression of CD45 by the cell types being analyzed. For the most part, this is not an issue when labeling mature hematopoietic cells. However, some progenitor stages (i.e., immature B lymphocytes) can express lower levels of CD45 thus reducing the sensitivity of the assay. In this case, it is important to pre-gate ex vivo stained CD45⁺ cells before evaluating CD45-PE (r.o.) labeling.

In addition, this protocol utilizes the r.o. sinus for injection of CD45-PE antibodies. Performing this step under anesthesia allows for relatively easy injection and delivery of the payload. However, it is considered invasive and discouraged in some facilities. Alternatively, tail vein injection can be performed; however, this method requires direct heating of mice to dilate the tail vein as well as extensive training to master.

Troubleshooting

Problem 1

Failed CD45-PE antibody labeling of blood cells upon analysis (observed while running samples on flow cytometer or analyzing samples in FlowJo, related to before you begin steps 2–3, step-by-step method details step 3).

Potential solution

• •Confirm calculations used to prepare CD45-PE antibodies for injection. A shift in decimal position could reduce the amount injected by 10-fold.
• •Inability to deliver the antibody solution into the r.o. sinus can result in failed labeling. However, this is usually evident at the time of injection and can be corrected via injection into the opposite sinus.
• •Perform ex vivo control stain of samples from PBS treated (or completely unrelated) mice using another PE-conjugated antibody that is pre-validated in your hands. A positive signal will confirm that your flow cytometer is configured properly and functioning normally.

Problem 2

Blood clots following cardiac puncture harvest (related to step-by-step method details steps 9–10).

Potential solution

• •This is likely due to not using an anti-coagulant during collection. Confirm that you are using blood collection tubes that are EDTA or heparin lined. Also, confirm that these tubes are within their expiration date.
• •Syringes which are already lined with anticoagulants can be utilized for initial blood collection (https://www.sai-infusion.com/products/1ml-treated-blood-collection-syringes?variant=14522303119415).

Problem 3

Hemolysis is observed following transfer of blood from syringe to BD Vacutainer tube (related to step-by-step method details step 10).

Potential solution

• •This may be due to excessive pressure when “flushing” collected blood cells, in particular red blood cells, through the syringe needle and into the BD Vacutainer tube. To reduce this occurrence, substitute a larger bore needle (i.e., 25G) for blood collection.

Problem 4

Failed CD45-APC ex vivo staining of cells upon analysis (observed while running samples on flow cytometer or analyzing samples in FlowJo, related to step-by-step method details steps 21–27).

Potential solution

• •If not properly titrated, this could result from competition for antibody binding epitopes when using the same clone for r.o. labeling as well as ex vivo staining. To resolve this issue, different CD45 antibody clones can be used. For example, r.o. labeling can be performed with CD45 antibody clone 30-F11 while ex vivo staining could use clone I3/2.3 (available from BioLegend, BD Biosciences and Thermo Fisher Scientific).

Problem 5

Insufficient ASC events collected for FlowJo analysis (observed while analyzing samples in FlowJo, related to step-by-step method details steps 21–27). ASCs are extremely rare and consist of as little as 0.01% of the total population of an organ.

Potential solution

• •Increase the number of cells per sample/organ that are stained and analyzed on the flow cytometer. This protocol uses 5 × 10⁶ cell per stain; however, this number can be increased to allow for more ASC events to be collected.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Peter Dion Pioli (peter.pioli@usask.ca).

Materials availability

This study did not generate new unique reagents.

Data and code availability

Data presented here are part of a larger dataset that was recently published by Pioli et al., 2023.¹ Original data are available from the corresponding author upon request.