Protocol for gene knockdown using siRNA in primary cultured neonatal murine microglia

Summary

In this protocol, we describe the small interfering RNA (siRNA)-mediated gene knockdown in primary mouse microglia, providing an approach to investigate functions such as phagocytosis and chemotaxis. The approach includes siRNA design, establishment of mixed glial cultures, microglia isolation, and siRNA transfection. Validation of knockdown efficacy employs quantitative immunoblot analysis. This technique empowers the investigation of specific molecular and cellular functions within the intricate microenvironment of the brain, comprising diverse cell types.

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

Graphical abstract

Highlights

• •Preparation of mouse primary microglia optimized for siRNA-mediated gene knockdown
• •Depicts efficient siRNA transfection and quantification via immunoblotting
• •Applicable for an array of subsequent assays like phagocytosis and chemotaxis

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

In this protocol, we describe the small interfering RNA (siRNA)-mediated gene knockdown in primary mouse microglia, providing an approach to investigate functions such as phagocytosis and chemotaxis. The approach includes siRNA design, establishment of mixed glial cultures, microglia isolation, and siRNA transfection. Validation of knockdown efficacy employs quantitative immunoblot analysis. This technique empowers the investigation of specific molecular and cellular functions within the intricate microenvironment of the brain, comprising diverse cell types.

Before you begin

Primary microglia are microglial cells isolated directly from living brains for cultivation. Used in combination with gene-knockdown techniques, primary microglia offer valuable insights into the intricate associations between specific molecules, signaling pathways, and cellular functions that are challenging to directly investigate in vivo.

In our prior publication,¹ we investigated the pathological roles of Alzheimer disease (AD) risk genes, notably TREM2, TYROBP, and INPP5D. By knocking down these genes in primary microglia, we investigated their effects on microglial functions, such as chemotaxis and cell adhesion. The present article provides an in-depth outline of our gene knockdown protocol, which is highly versatile and can be used for any gene and for the analysis of diverse cellular phenotypes.

Institutional permissions

All experiments were conducted in accordance with the protocols approved by the Institutional Animal Care Committee of the Graduate School of Pharmaceutical Sciences at the University of Tokyo (protocol no. P27-6, P2-2, and P5-5). It is important to note that ethical guidelines and standards vary across countries and institutions. Before beginning your experiments, ensure that you have the necessary approval from your institution’s relevant committee.

Preparation of siRNAs

Timing: 1 h

Small interfering RNAs (siRNAs) are 21–25 base pair double-stranded RNAs with dinucleotide 3′ overhangs at both ends. When transfected into cultured cells, siRNAs induce RNA interference (RNAi) and suppress the expression of complementary sequences. You can obtain siRNAs either from commercial sources or by custom designing based on the target gene sequence.

• 1.
  siRNA preparation.

  • a.
    Choose pre-designed siRNAs from commercial sources like Dharmacon.
  • b.
    If pre-designed siRNAs are not available, design custom siRNA sequences. Use online tools such as siDirect (http://sidirect2.rnai.jp/)^2,3,4 and then have the designed sequences synthesized by vendors such as GeneDesign, Inc.

Note: We recommend to select siRNAs that are labeled as “validated”, meaning they have been experimentally tested and confirmed for their efficacy in knocking down the target gene.

Note: Due to variability in knockdown efficiency and potential off-target effects, design two or three distinct siRNA sequences for each target gene. Such off-target interactions can lead to reduced post-transfection cell survival. Multiple sequences help mitigate the risk of non-specific binding and off-target interactions.

Note: Use appropriate control siRNAs in experiments to distinguish sequence-specific silencing from any off-target effects. For example, we use non-targeting siRNAs that are confirmed by BLAST to have at least 4 mismatches to mouse transcripts (the specific sequences used in our previous study¹ are listed in Table S1). Another option is to use siRNAs that target irrelevant genes in your experimental context, such as luciferase or green fluorescent protein (GFP). Finally, scrambled versions of siRNAs with the same nucleotide composition but in a random order can also be used as controls. They control for any non-specific effects arising from the introduction of exogenous siRNA.

• 2.
  Preparation of the working stock.

  • a.
    Dissolve powdered siRNA in RNase-free water to make a 50 μM stock solution, and store it at −80°C.
  • b.
    Dilute the stock solution to 10 μM using RNase-free water.
  • c.
    When preparing a pool of siRNAs with different sequences targeting the same gene, mix equal volumes of 10 μM siRNAs.

Note: When reconstituting powdered siRNA, it is recommended to avoid immediate pipetting after adding water, as the siRNA powder can easily adhere to pipette tips. Instead, gently add RNase-free water and leave the tube for an hour with slight agitation at 4°C to ensure uniform hydration. Once hydrated, the siRNA can be subjected to vortexing. Ensure thorough vortexing to guarantee complete dissolution of the siRNA.

Preparation of L929 cell-conditioned medium

Timing: 9 days

Primary microglia require macrophage colony-stimulating factor (M-CSF) for growth and survival. In mixed glial cultures, the necessary M-CSF is provided by astrocytes. However, isolated microglia lack this endogenous source and must receive supplemental M-CSF. The mouse fibroblast L929 cell line secretes M-CSF into its conditioned medium (LCM), which is an efficient and cost-effective replacement. However, although LCM can substitute for M-CSF, it contains additional secreted factors that could skew outcomes.⁵ Researchers should consider experimental endpoints when choosing recombinant M-CSF versus LCM. We typically supplement isolated microglia media with 10% (v/v) LCM as the M-CSF source.^5,6,7

• 3.Seeding (Day 1).

Seed L929 cells in DMEM/FBS/PS medium at a density of 4.7 × 10⁵ cells, and a volume of 15 mL per 75-cm² flask.

• 4.Medium addition (Day 2).

Add an additional 40 mL of DMEM/FBS/PS to each flask, bringing the total volume to 55 mL.

• 5.Medium collection (Day 9).

After a week of culturing, collect the medium. Subsequently, filter the medium through a 0.22-μm filter unit, aliquot 10 mL each into 15-mL tubes, and store at −20°C or below until use. The medium is stable for at least 2–3 months under this storage condition.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit monoclonal anti-Phospho-Akt (Ser473) (D9E) (1:2,000 dilution)	Cell Signaling Technology	Cat#4060; RRID: AB_231504
Rabbit monoclonal anti-Akt (pan) (C67E7) (1:1,000 dilution)	Cell Signaling Technology	Cat#4691; RRID: AB_915783
Rabbit polyclonal anti-Phospho-PLC-gamma-2 (Tyr1217) (1:1,000 dilution)	Cell Signaling Technology	Cat#3871; RRID: AB_229954
Rabbit polyclonal anti-PLC-gamma-2 (1:1,000 dilution)	Cell Signaling Technology	Cat#3872; RRID: AB_229958
Rabbit monoclonal anti-TREM2 (E7P8J) (1:1,000 dilution)	Cell Signaling Technology	Cat#76765; RRID: AB_2799888
Rabbit monoclonal anti-DAP12 (D7G1X) (1:1,000 dilution)	Cell Signaling Technology	Cat#12492; RRID: AB_272112
Mouse monoclonal anti-SHIP1 (P1C1) (1:200 dilution)	Santa Cruz Biotechnology	Cat#sc-8425; RRID: AB_628250
Mouse monoclonal anti-α-Tubulin (clone DM1A) (1:2,000 dilution)	Sigma-Aldrich	Cat#T9026; RRID: AB_477593
Mouse monoclonal anti-GAPDH (6C5) (1:5,000 dilution)	Santa Cruz Biotechnology	Cat#sc-32233; RRID: AB_627679
Goat anti-rabbit peroxidase (1:4,000–8,000 dilution)	Jackson ImmunoResearch	Cat#111-035-144; RRID: AB_2307391
Goat anti-mouse peroxidase (1:4,000–8,000 dilution)	Jackson ImmunoResearch	Cat#115-035-003; RRID: AB_10015289
CD16/32 antibody (final 0.5 μg/mL)	Miltenyi Biotec	Cat#130-092-574; RRID: AB_871624
Alexa Fluor 647 anti-mouse CD45 (1:100 dilution)	BioLegend	Cat#103124; RRID: AB_493533
FITC anti-mouse CD11b antibody, REAfinity (1:50 dilution)	Miltenyi Biotec	Cat#130-113-805; RRID: AB_2726325
Chemicals, peptides, and recombinant proteins
Opti-MEM I reduced serum medium, no phenol red	Gibco	Cat#11058021
Lipofectamine RNAiMAX transfection reagent	Thermo Fisher Scientific	Cat#13778500
D-MEM (Dulbecco’s modified Eagle’s medium) (high glucose) with L-glutamine and phenol red	FUJIFILM Wako	Cat#044-29765
HBSS (Hank’s balanced salt solution) (−) without phenol red	FUJIFILM Wako	Cat#085-09355
Sodium chloride (NaCl)	FUJIFILM Wako	Cat#196-01671; CAS: 7647-14-5
Disodium hydrogenphosphate dodecahydrate (Na2HPO4 12H2O)	FUJIFILM Wako	Cat#196-02835; CAS: 10039-32-4
Potassium chloride (KCl)	FUJIFILM Wako	Cat#163-03545; CAS: 7447-40-7
Potassium dihydrogen phosphate (KH2PO4)	FUJIFILM Wako	Cat#169-04245; CAS: 7778-77-0
Tris(hydroxymethyl) aminomethane (C4H11NO3)	FUJIFILM Wako	Cat#207-06275; CAS: 77-86-1
Hydrochloric acid (HCl)	FUJIFILM Wako	Cat#080-01066
Tween 20	Sigma-Aldrich	Cat#P1379-500ML; CAS: 9005-64-5
MgSO4	KANTO	Cat#58042-17; CAS: 10034-99-8
CaCl2	KANTO	Cat#58001-17; CAS: 10035-04-8
DNase I	NIPPON GENE	Cat#311-08081
Trypsin (2.5%), no phenol red	Gibco	Cat#15090046
Penicillin-Streptomycin solution (×100)	FUJIFILM Wako	Cat#168-23191
Distilled water, deionized, sterile	NIPPON GENE	Cat#318-90105
Fetal bovine serum (FBS) standard, origin South America	Cosmo Bio Co., Ltd.	Cat#CCP-FBS-BR-500
RIPA buffer	FUJIFILM Wako	Cat#188-02453
cOmplete protease inhibitor cocktail	Roche	Cat#11836145001
PhosSTOP	Roche	Cat#4906837001
Precision Plus Protein Dual Xtra Standards	Bio-Rad	Cat#1610377
0.5 M EDTA (pH 8.0)	NIPPON GENE	Cat#311-90075; CAS: 6381-92-6
2-Mercaptoethanol	Sigma-Aldrich	Cat#M6250; CAS: 60-24-2
Sodium dodecyl sulfate (SDS)	FUJIFILM Wako	Cat#191-07145; CAS: 151-21-3
Glycerol	FUJIFILM Wako	Cat#075-00616; CAS: 56-81-5
Brilliant green	FUJIFILM Wako	Cat#021-02352; CAS: 633-03-4
Coomassie brilliant blue G-250	Nacalai Tesque	Cat#09409-42; CAS: 6104-58-1
Difco skim milk	BD	Cat#232100
Albumin, from bovine serum, fraction V pH 7.0	FUJIFILM Wako	Cat#013-27054
Propidium iodide	FUJIFILM Wako	Cat#169-26281
Critical commercial assays
BCA Protein Assay Kit	Takara	Cat#T9300A
ImmunoStar reagents	FUJIFILM Wako	Cat#291-55203
Experimental models: Cell lines
Mouse L-929 cells	ECACC	RRID: CVCL_0462
Experimental models: Organisms/strains
Mouse: C57BL/6JJmsSlc (pregnant females; 10–11 week old)	Japan SLC, Inc.	RRID: MGI:5488963
Oligonucleotides
siRNAs, see Table S1		N/A
Software and algorithms
Fiji (1.53c)	ImageJ	RRID: SCR_003070
RStudio (version 1.3.1056)	RStudio, PBC	RRID: SCR_000432
R (version 4.0.2)	R Foundation for Statistical Computing	RRID: SCR_001905
Other
Tweezers Dumont INOX No.7	Dumont	Cat#0102-7B-PO
Tweezers Dumont INOX No.5	Dumont	Cat#0108-5-PO
100-μm cell strainer	AS ONE	Cat#VCS-100
MultiWell plate for suspension culture 6F with lid	Sumitomo Bakelite Co., Ltd.	Cat#MS-8006RZ
100-mm tissue culture dish	IWAKI	Cat#3020-100
20-mL syringe	Terumo	Cat#SS-20ESZ
Stericup quick release-GV sterile vacuum filtration system, 500 mL, 0.22 μm, PVDF membrane	Merck Millipore	Cat#S2GVU05RE
0.22-μm polyvinylidene fluoride (PVDF) filter	Merck Millipore	Cat#SLGV033RS
Nunc EasYFlask 75 cm2	Thermo Fisher Scientific	Cat#156472
Multi-application cell sorter MA900	Sony	N/A
ImageQuant LAS 4000	Cytiva	N/A
Leica S8 APO unique Greenough stereo microscope with apochromatic optics	Leica Microsystems	Cat#S8 APO

Materials and equipment

DMEM/FBS/PS

Reagent	Final concentration	Amount
Dulbecco’s modified Eagle medium (DMEM)	N/A	500 mL
Fetal bovine serum (FBS)	10% (v/v)	50 mL
100× Penicillin-streptomycin solution	1×	5 mL
Total		555 mL

Store at 4°C for up to 1 month.

Note: We routinely subject FBS to heat-inactivation before use. To do this, thaw the frozen FBS bottle for 8–24 h at 4°C. Once completely thawed, immerse the bottle in a water bath heated to 56°C, and let it undergo inactivation for 30 min. After thorough mixing, dispense the mixture into 50-mL aliquots and store them at −20°C or below.

25× Dulbecco’s phosphate-buffered saline (DPBS)

Reagent	Final concentration	Amount
NaCl	3.75 M	219.15 g
Na2HPO4 12H2O	200 mM	71.63 g
KCl	67.5 mM	5.03 g
KH2PO4	37.5 mM	5.10 g
Double-distilled H2O	N/A	Fill up to 1 L
Total		1 L

Store at room temperature; stable for 1 year.

1× DPBS

Reagent	Final concentration	Amount
25× DPBS	1×	40 mL
Double-distilled H2O	N/A	Fill up to 1 L
Total		1 L

Autoclave (121°C, 15 min) and store at 18°C–26°C; stable for 1 year.

10× Tris-buffered saline

Reagent	Final concentration	Amount
Tris(hydroxymethyl)aminomethane	500 mM	181.71 g
NaCl	1.5 M	263.25 g
HCl	N/A	Until pH reaches 7.6
Double-distilled H2O	N/A	Fill up to 3 L
Total		3 L

Store at 18°C–26°C; stable for 1 year.

0.1% (v/v) Tween 20 in Tris-buffered saline (TBST)

Reagent	Final concentration	Amount
10× Tris-buffered saline	1×	600 mL
Tween 20	0.1% (v/v)	6 mL
Double-distilled H2O	N/A	Fill up to 6 L
Total		6 L

Store at 18°C–26°C; stable for 1 year.

2× enzyme solution

Reagent	Final concentration	Amount
Hanks’ balanced salt solution (HBSS) (−)	N/A	4.5 mL
Trypsin	0.25%	0.5 mL
MgSO4	1.6 mM	10 μL
CaCl2	3.7 mM	10 μL
Deoxyribonuclease I	3–5 units/mL
Total		5 mL

Prepare immediately before use.

Lysis buffer

Reagent	Final concentration	Amount
RIPA buffer	N/A	50 mL
cOmplete Protease Inhibitor Cocktail	N/A	1 tablet
Total		50 mL

Prepare immediately before use.

5× sample buffer

Reagent	Final concentration	Amount
Sodium dodecyl sulfate (SDS)	10%	4 g
1 M Tris-HCl, pH 6.8	0.4 M	16 mL
Glycerol	50%	20 mL
1% (w/v) Brilliant green	0.0125%	0.50 mL
1% (w/v) Coomassie brilliant blue G-250	0.03125%	1.25 mL
Double-distilled H2O	N/A	Fill up to 40 mL
Total		40 mL

Store at 18°C–26°C; stable for 1 year.

Step-by-step method details

Isolation of microglia

Timing: 12 days (2 h for steps 1–4)

This microglia isolation protocol is based on the foundational techniques established by Giulian and Baker⁸ and Frei et al.,⁹ while omitting the time-consuming mechanical shaking and sequential replating steps. Single-cell suspensions are prepared from the brains of newborn mice, and these mixed glial cells are cultured with proliferating astrocytes forming a feeder layer. Once sufficient astrocyte coverage is achieved, loosely adherent microglia begin to expand. At this stage, pure microglia are effectively detached simply by vigorously tapping the flasks by hand. Microglia isolated through similar protocols actively release various cytokines in response to stimuli such as lipopolysaccharide or other factors.^10,11 This ability makes them a useful tool for studying the role of microglia in neuroinflammation.

• 1.
  Sterilization of instruments.

  • a.
    Sterilize a stereomicroscope by exposing it to ultraviolet light.
  • b.
    Disinfect scissors, forceps, and spatulas by soaking them in 70% ethanol.
  • c.
    Have ready: several 10-cm dishes filled with cold HBSS(−), 20-mL syringes, a 0.22-μm polyvinylidene fluoride (PVDF) filter, a 100-μm cell strainer, and 75-cm² flasks.
• 2.Reagent making.

Prepare 2× enzyme solution and sterilize it by passing it through a 0.22-μm PVDF filter.

• 3.
  Dissection.

  • a.
    Immobilize neonatal (postnatal day 1–3) mice on ice.
  • b.
    Decapitate the mice with sharp scissors and place their heads in a 10-cm dish with ice-cold HBSS (−).
  • c.
    Use fine forceps to peel away the scalp, make an incision along the midline of the skull, and then remove the skull bones laterally, exposing the brain.
  • d.
    Using a spatula, gently transfer the brains to a new dish with ice-cold HBSS (−).
  • e.
    Using fine forceps, remove any visible meningeal membranes under a stereomicroscope.
  • f.
    Transfer the cleaned brains to a 50-mL tube with 5 mL of fresh HBSS (−) on ice. Pool up to 6–8 brains together.

Note: For optimal tissue preservation, it is advisable to decapitate no more than 4 to 5 mice simultaneously. If you are unfamiliar with the procedure, start with one mouse.

Note: For details on the removal of skin, skull, and meningeal membranes, we recommend that readers refer to Bronstein et al.¹² This reference demonstrates the technique with an excellent video showing the entire tissue extraction process under a stereomicroscope (see the segment from 2:05–4:10).

• 4.
  Cell suspension.

  • a.
    After dissecting all the brains, combine them with the 2× enzyme solution from step 2, and incubate at 37°C for 5 min in a water bath.
  • b.
    Gently pipette the tissue using a 10-mL serological pipette fitted with a 100–1000-μL pipette tip at the end. Incubate again for 5 min at 37°C.
  • c.
    Repeat the pipetting and incubation process two more times.
  • d.
    Pipette the suspension using a 10-mL serological pipette fitted with a 20–200-μL pipette tip to dissociate cell aggregates.
  • e.
    Add 30 mL of DMEM/FBS/PS to stop the enzyme reaction. Filter the suspension through a 100-μm cell strainer into a fresh 50-mL tube.
  • f.
    Centrifuge at 740 × g for 5 min, discard the supernatant, and resuspend the cells in DMEM/FBS/PS.
  • g.
    Seed the cells in 75-cm² flasks at a density of 1 brain in 15 mL medium per flask. Define this day as day 0 in vitro (DIV).
• 5.
  Mixed glial culture and microglia isolation.

  • a.
    Change the medium on 3 and 7 DIV. Expect astrocyte adhesion between 1 and 3 DIV and confluency by about 7 DIV.
  • b.
    Observe microglial proliferation starting from 7 DIV, with some cells detaching from the astrocytic layer and floating in the culture medium. Once substantial floating cells appear (usually by 11 DIV), proceed with the following steps.
  • c.
    Secure the flask cap tightly. Vigorously tap the flask approximately 50 times by hand to detach only the loosely adherent cells.
  • d.
    Collect the supernatant and centrifuge at 740 × g for 5 min.
  • e.
    Discard the supernatant, resuspend the cell pellet in 10% LCM, and seed at 3 × 10⁵ cells/well in 2 mL media for a 6-well plate.

    Note: We typically avoid changing the medium at 1 DIV. Whereas some non-adherent cell debris can be seen, changing the media at this early time point tends to disrupt the fragile adhering astrocytes, and the microglia that have begun to attach.

    Note: Typically, you can obtain 1 × 10⁶ microglial cells from a single flask. If cell proliferation is slow, refer to troubleshooting 1. If the yield is insufficient, refer to troubleshooting 2.

    Note: When collecting microglia, note that they tend to adhere to the inner walls of the tubes. Keeping the suspension on ice can help reduce cell loss.

    Note: For seeding primary microglia, consider using low-adherence plates, such as Sumitomo Bakelite’s multiwell suspension culture plates. In our experience, these plates facilitate higher cell yields than tissue culture-treated plates, based on bicinchoninic acid (BCA) protein quantification. However, researchers should optimize the protocol by comparing plate types, because adhesion may affect phenotypes.

    Optional: Determination of microglia purity by flow cytometry

    To determine the purity of microglia (in our case, typically exceeding 95%), we assess the population of cells doubly positive for the CD11B and CD45 markers using flow cytometry (Figure 1). Follow the protocol below.

    • i.
      Plate primary microglia on low-adherence 6-well plates at a density of 3 × 10⁵ cells/well. Use 2 mL of 10% LCM and culture for 48 h.
    • ii.
      Wash the cells with DPBS and treat them with 0.125% trypsin and 1 mM EDTA at 37°C for 5 min.
    • iii.
      Add DPBS containing 2% FBS to each well to stop the enzyme reaction.
    • iv.
      Gently pipette the cells to collect and transfer them to a fresh tube.
    • v.
      Spin the cells down at 300 × g for 5 min. Discard the supernatant and resuspend the cells in DPBS containing 1% BSA.
    • vi.
      After another centrifugation, resuspend the cell pellet in DPBS containing 0.5 μg/mL of CD16/32 antibody to block Fc receptors on microglia. Place on ice for 10 min.
    • vii.
      Incubate the cell suspension with Alexa Fluor 647 anti-mouse CD45 antibody and anti-mouse CD11B antibody on ice for 30 min.
    • viii.
      Spin down the cells at 300 × g for 5 min, discard the supernatant, and resuspend the cell pellet in DPBS containing 1% BSA.
    • ix.
      Add 1 μg/mL propidium iodide to stain dead cells immediately before measurement.
    • x.
      Analyze the cell population using a flow cytometer.

Figure 1.

Flow cytometry analysis of primary microglia

(A) A plot of FSC-A vs. SSC-A illustrates gating to exclude debris and to identify intact cells.

(B) A plot of FSC-H vs. FSC-W showing gating to select singlets.

(C) A histogram of cells stained with propidium iodide, indicating viable cells that excluded the dye.

(D) A plot of FITC (CD11B) vs. Alexa Fluor 647 (CD45) with gating showing the double-positive microglial cell population.

(E) A similar plot as in D but without antibody staining, used as a negative control. The typical purity of viable microglia obtained by this protocol is more than 95%.

siRNA knockdown

Timing: 5 days

In this procedure, isolated microglia are transfected with siRNA. Before proceeding, ensure that the primary microglia are healthy and firmly adhered to the plate; cells in suboptimal conditions may not survive the transfection process.

• 6.Transfection of siRNA (Day 1).
  Typically, primary microglia are transfected with siRNA 2 or 3 days after seeding to ensure their optimal adherence. The following is our protocol for siRNA transfection using Lipofectamine RNAiMAX reagent (refer to the manufacturer’s instruction for additional details). This protocol is specifically designed for a 6-well plate format.

  • a.
    Pipette 150 μL of Opti-MEM medium into an Eppendorf tube. Add 3 μL of 10 μM siRNA working stock and vortex briefly.
  • b.
    In a separate tube, dilute 9 μL of Lipofectamine RNAiMAX reagent in 150 μL of Opti-MEM medium. Mix thoroughly by vortexing for several seconds.
  • c.
    Combine the solutions from steps a and b, vortex gently, and incubate at 18°C–26°C for 5 min.
  • d.
    Remove the cell plate from the incubator. Dropwise, add 250 μL of the RNA-lipid complex prepared in step c, ensuring an even distribution. Gently shake the plate for a uniform distribution and then return it to the incubator.

    Note: Adjust the volumes accordingly when using different well formats based on the bottom area of the well. When transfecting multiple wells or using different siRNAs simultaneously, prepare a master mix of the RNAiMAX diluent in one tube for all samples. Optimize the amount of siRNA based on the knockdown efficiency of the target gene for each experiment.

    Note: If cell viability is low after siRNA transfection, refer to troubleshooting 3.

• 7.Changing the medium (Day 2).

Twenty-four hours after transfection, replace the medium with 2 mL 10% LCM medium per well.

Note: This step enhances cell viability without reducing the knockdown efficiency. Because transfected cells can detach easily, add the medium gently to prevent detachment.

• 8.Cell analysis (Day 5).

Seventy-two hours post-transfection is typically optimal for observing knockdown efficiency. Assess cell condition and proceed with the following steps.

Optional: For alternate applications, such as phagocytosis or chemotaxis assays, consider trypsinizing cells about 48 h post-transfection. These cells can then be re-seeded onto suitable culture vessels, such as plates, dishes, or migration chambers. Typically, after 24 h, once the cells have firmly attached to the vessel, they are ready for subsequent analyses.

Lysate preparation and protein analysis by immunoblotting

Timing: 1 h (for steps 9–12), 2 days (for step 13)

This procedure involves the preparation of cell lysates, followed by the analysis of protein expression.

• 9.Preparation of lysis buffer.

Prepare the lysis buffer, ensuring it is well-chilled on ice before use.

Note: Do not add reducing reagents, as they interfere with the BCA protein assay.

Optional: To detect phosphoproteins, add phosphatase inhibitors, such as PhosSTOP.

• 10.
  Sample collection.

  • a.
    Place the plate on ice. Wash the samples twice with ice-cold phosphate-buffered saline (PBS).
  • b.
    Add 100 μL of lysis buffer to each well (for a 6-well format). Incubate the samples on ice for 5 min.
  • c.
    Using a cell scraper, transfer the lysates to pre-chilled microcentrifuge tubes and place the tubes on ice.

Note: To prevent protein degradation, keep the samples on ice.

• 11.BCA assay.

Quantify proteins using a BCA protein assay kit available from vendors, such as Takara and Thermo Fisher Scientific.

• 12.
  Preparation of immunoblotting samples.

  • a.
    Prepare 5× sample buffer.
  • b.
    To the sample, add the 5× sample buffer to a final 1× concentration, and 2-mercaptoethanol to a final concentration of 1% (v/v) in the sample to reduce disulfide bonds.
  • c.
    Incubate samples at 100°C for 5 min to denature polypeptides.

Pause point: You can store the samples at −20°C or below at this step. Thaw thoroughly before use and avoid repeated freeze-thaw cycles.

• 13.
  Immunoblotting.

  • a.
    Perform SDS polyacrylamide gel electrophoresis using either Tris-glycine or Tris-tricine gels, choosing the appropriate acrylamide concentration based on the molecular weight of the proteins of interest.
  • b.
    Electroblot the separated proteins onto PVDF membranes.
  • c.
    Block non-specific binding on the membranes with 5% skim milk or 3% BSA (for phospho-specific antibodies), both prepared in 0.1% (v/v) Tween 20 in TBST. Incubate for 1 h at 18°C–26°C.
  • d.
    Wash the membranes 3 times with TBST.
  • e.
    Incubate the membranes with the primary antibody diluted in TBST supplemented with 0.02% NaN₃ for 8–24 h at 4°C.
  • f.
    Wash the membranes 3 times with TBST.
  • g.
    Incubate the membranes with horseradish peroxidase-conjugated secondary antibody, suitably diluted in TBST, for 1 h at 18°C–26°C.
  • h.
    Wash the membranes 3 times with TBST.
  • i.
    Apply an enhanced chemiluminescence reagent, such as ImmunoStar, to the membrane. Visualize and capture the signals using a LAS-4000 LuminoImager.

Expected outcomes

Following this protocol, the anticipated yield is approximately 1 × 10⁶ microglia per pup, with a purity exceeding 95%. This assessment is based on flow cytometry, which identifies the CD11B+/CD45+ cell population, as depicted in Figure 1.

When primary microglia are treated with siRNA against Trem2, Tyrobp, or Inpp5d, we typically observe that their expression level is substantially reduced, as determined by immunoblotting shown in Figures 2 (and referenced as Figure 7A, B in paper¹) and 3. If the knockdown efficiency is low, refer to troubleshooting 4.

Figure 2.

Effects of Trem2, Tyrobp, or Inpp5d knockdown on AKT and PLCγ2 phosphorylation in primary microglia

(A) Representative immunoblots depicting the protein levels of p-AKT and p-PLCγ2 relative to the total levels of AKT and PLCγ2, respectively (Figure 7A and B of the reference paper¹).

(B) Quantitative analysis of phosphorylated protein levels, with p-AKT (n = 15) and p-PLCγ2 (n = 9) levels normalized to total AKT and PLCγ2 levels, respectively.

Data represent the mean ± SEM. ns: not significant (p > 0.05), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by the repeated measures one-way ANOVA, followed by Tukey’s multiple comparisons.

Figure 3.

siRNA-mediated knockdown of Inpp5d in primary microglia

(A) Representative immunoblots for INPP5D and GAPDH proteins 72 h after no transfection, transfection with non-targeting control siRNA, or Inpp5d-targeting siRNA.

(B) Quantification of INPP5D knockdown efficiency. The INPP5D band intensity was normalized first to the corresponding GAPDH intensity. Then, the ratio of INPP5D/GAPDH in the non-transfected sample was set at 1 for each independent experiment. Data represent the mean ± SEM (n = 3). ∗∗∗P < 0.001 by the unpaired, two-tailed Student’s t-test.

After siRNA treatment, these primary microglia are suitable for various downstream analyses, including assays for phagocytosis and chemotaxis, as well as immunoblot analysis, as illustrated in Figure 2. We observed that knockdown of Trem2, Tyrobp, or Inpp5d differentially affects the phosphorylation levels of AKT and PLCγ2.

Quantification and statistical analysis

In a typical analysis, cell groups treated with siRNAs targeting the gene(s) of interest are compared with those treated with control siRNA, with both being run on the same gel.

To minimize variability in band intensity quantification, samples should be diluted to achieve equivalent total protein concentrations across all lysates, based on BCA assay results. Loading equal volumes of lysate helps account for pipetting errors and improves detection accuracy across samples.

After immunoblotting, the band intensity of each lane can be quantified using ImageJ software. For accurate quantification, it is crucial to account for possible variations in protein loading during immunoblotting. Loading control normalization is achieved by normalizing the band intensity of each sample to a loading control, such as α-tubulin or GAPDH. This normalization ensures that the observed changes in protein expression genuinely result from the knockdown itself. When analyzing protein phosphorylation, as exemplified in Figure 2, samples are probed with two antibodies—one phospho-specific to detect the phosphorylated form and another pan-antibody that detects the total target regardless of the phosphorylation. By calculating the ratio of these intensities, we can obtain normalized quantitative values representing the levels of the phosphorylation.

In addition, we routinely analyze lysates from non-transfected cells simultaneously with our test samples. By standardizing the expression ratio of all bands to this control sample as 1 for each distinct trial, we control for exposure differences between immunoblots. This enables the accurate comparison of siRNA-transfection conditions across replicate experiments. Furthermore, it is helpful for detecting off-target effects of siRNA. If there are functional differences between non-target siRNA-transfected cells and non-transfected cells, refer to troubleshooting 5.

Finally, data interpretation should be performed using suitable statistical methods. For adequate analysis, it is essential to replicate the same experiments at least 3 times; however, the exact number of required replicates can vary based on the degree of difference between samples or the total number of groups being compared. For comparisons between 2 groups, the t-test is typically appropriate. For multiple group comparisons, an ANOVA followed by a post-hoc test, such as the Tukey’s or Dunnett’s test, is recommended. Using these rigorous methods ensures the credibility and reproducibility of the findings.

Limitations

There are several limitations to consider when using the primary microglia obtained through this protocol. It has been reported that these microglia can display variations in both gene expression and morphology compared with in vivo microglia.^13,14 Therefore, the outcomes should be interpreted with caution. For example, the transcriptional response of microglia to amyloid β, a peptide linked to the pathogenesis of AD, may differ between primary microglia and in vivo microglia in an AD mouse model.¹⁵ Nevertheless, primary microglia are a crucial tool for studying molecular mechanisms that are challenging to investigate in vivo.

Whereas this protocol is optimized for neonatal mice, there may be instances, such as in aging research, in which microglia from adult mice are required. Note that the isolation and culturing of microglia from adult mice is challenging.¹²

Primary microglia readily accommodate siRNA transfection, resulting in high knockdown efficiency, as demonstrated in this protocol. On the other hand, transfection of DNA constructs is difficult in primary microglia, owing to potential DNA toxicity. For gene overexpression, the use of a recombinant lentiviral or adeno-associated viral vector is recommended. Through our experiences using these vectors, we have found satisfactory transfection efficiency for small genes, such as that of GFP. However, larger gene inserts might require further optimization.

Whereas siRNA-mediated knockdown is effective, it is vital to understand that such knockdown is both transient and not complete. In some cases, residual or rebounding mRNA/protein levels can obscure the intended knockdown effect. It is hence advisable to verify the knockdown efficiency throughout the experiment.

Troubleshooting

Problem 1

Cell proliferation is slow in a mixed glial culture (related to Step 5).

Potential solution

• •Ensure that the lid of 75-cm² flask is loosened sufficiently to enable adequate CO₂ exchange. Primary cells are sensitive to impaired gas diffusion, which can slow their growth.
• •Check whether astrocytes have fully covered the flask surface at about 7 DIV. Adequate astrocyte adhesion and expansion are crucial factors for microglial proliferation. If astrocyte attachment appears incomplete, consider extending the culture time for several days to help establish the astrocyte feeder layer.

Problem 2

Insufficient yield of primary microglia (related to Step 5).

Potential solution

• •A suboptimal quality of FBS in the culture medium might affect the yield.
• •Maintain the CO₂ concentration at 5% during culture because any reduction can compromise microglial viability.
• •Consider extending the mixed glial culturing duration. Change the medium regularly every 3–4 days to support cell proliferation. However, once microglia begin to proliferate and float, continue culturing without changing the medium for up to 6 days. Frequent medium changes can not only lead to the loss of floating cells but also reduce the concentration of M-CSF in the medium.

Problem 3

Cells are prone to death after knockdown (related to Step 6).

Potential solution

• •Try alternative siRNA sequences.
• •Use a freshly prepared siRNA stock.
• •Knockdown of certain genes may cause cell death or cell cycle arrest, resulting in an insufficient number of cells for downstream analysis. In such scenarios, as observed in our Trem2 or Tyrobp knockdown experiments, it is advisable either to increase the initial seeding density of the primary microglia, or to seed cells in additional wells at the start of the experiment.

Problem 4

Low knockdown efficiency (related to Expected outcomes).

Potential solution

• •Try alternative siRNA sequences.
• •Ensure that the chosen siRNA sequence is not designed to target an intron.

Problem 5

Observed discrepancies between non-target siRNA-transfected cells and non-transfected cells, particularly in functional assays (related to quantification and statistical analysis).

Potential solution

• •This issue may be attributed to the off-target effects of non-target siRNA. Consider using an alternative siRNA sequence as the control.
• •Multiple freeze-thaw cycles of the siRNA might affect its efficacy. Prepare and use a fresh siRNA stock.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Taisuke Tomita (taisuke@mol.f.u-tokyo.ac.jp).

Technical contact

For specific questions and details regarding the technical aspects of the protocol, please address correspondence to the technical contact, Sho Takatori (takatori@mol.f.u-tokyo.ac.jp).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The published article includes all the datasets generated and analyzed in this study. https://www.sciencedirect.com/science/article/pii/S2589004223004522.

This study does not report an original code.

Thumbnails for the Graphical abstract were prepared using BioRender.com.