May 20, 2026
Primary murine microglia, neurons and co-cultures
- 1KU Leuven
Protocol Citation: Aisha Sati 2026. Primary murine microglia, neurons and co-cultures . protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6eyprgqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 24, 2026
Last Modified: May 26, 2026
Protocol Integer ID: 243996
Keywords: ASAPCRN, primary murine microglia, primary microglia, neuronal culture, transfections of sirna, methodology for polyamine supplementation, polyamine supplementation, neuron
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-024297
Abstract
This protocol details the methodology to establish primary murine microglia, neuronal cultures and co-cultures. The protocol also describes methodology for polyamine supplementation in vitro and transfections of siRNA in primary microglia.
Materials
Enzyme (20mL):
- 0.0064 g L-cysteine (Sigma)
- 20 mL DM
- 400 units papain (Worthington)
- Incubate 30’ at 37°C until solution is clear
- pH enzyme solution with 0.1N NaOH until it looks like DM again
- Syringe filter enzyme solution
Plating Media (50 mL):
- 5 mL horse serum (Invitrogen)
- 1 mL 1M glucose
- 0.1 mL 100x pen/strep (Invitrogen)
- 0.5 mL 100x glutamax (Invitrogen)
- 0.5 mL 100mM sodium pyruvate (Invitrogen)
- 43 mL MEM (Invitrogen)
Hippocampal Feeding Media (HFM) (50 mL):
- 1 mL B27 (Invitrogen)
- 0.6 mL 1M glucose
- 0.1 mL 100x pen/strep (Invitrogen)
- 0.125 mL 100x glutamax (Invitrogen)
- 1 μL 1.25M ß-mercapto-ethanol
- 48.2 mL Neurobasal-A (Invitrogen)
HBSS (500 mL):
- 50 mL 10x HBSS (Invitrogen)
- 1.25 mL 1M HEPES (pH 7.4)
- 15 mL 1M glucose
- 5 mL 100mM CaCl2
- 5 mL 100mM MgSO4
- 2 mL 1M NaHCO3
- 421.76 mL H2O; sterile filter solution; store at 4°C, use within 1 month
DMEM 10:10:1:
DMEM (Gibco,11965092)
10% horse serum (Gibco, 2605-088)
10% fetal bovine serum (Gibco, A5209501)
1% Penicillin/streptomycin (Sigma, P4458)
Protocol materials
poly-D-lysineMilliporeSigma
lamininSigma aldrich.com
ON-TARGETplus SMARTpool siRNA,Horizon DiscoveryCatalog #L-051693-01-0010
DharmaFECT transfection reagentHorizon DiscoveryCatalog #T-2005-01
HBSSInvitrogen
MEM,Invitrogen
DMEM,Gibco - Thermo Fisher ScientificCatalog #11965092
Penicillin/streptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4458
Horse SerumGibco - Thermo Fisher Scientific
FBSGibco - Thermo Fisher ScientificCatalog #A5209501
Trypan blueGibco - Thermo Fisher Scientific
LPS,Sigma aldrich.comCatalog #serotype 0111:B4
Spd
Neurobasal medium,Invitrogen
PFA
MitoTEMPO Sigma aldrich.comCatalog #SML0737
AMXT 1501
Aminoguanidine
Microglia culture
7h 50m
Coat 75 cm2 flasks and 12 well plates
add 10 mL of 5µg/ml µg/ml poly-D-lysineMilliporeSigma per flask, and 500ul per well of the 12-well plates containing round glass coverslips.
30m
Incubate at 37 °C for 02:00:00 . Wash 3x with mqH2O. Let dry after washes and store at -20 °C if not used immediately.
2h
Brain dissection, meninges removal and preparation of single cell suspension
3h
Sacrifice PND0-2 pups and wash the heads with sterile PBS
Dissect the brain in DMEM,Gibco - Thermo Fisher ScientificCatalog #11965092 , and carefully remove the meninges with fine foreceps, then removing the olfactory bulb and cerebellum,
Use a flame-polished glass pipette (approximately half the original diameter) to homogenize the tissue by pipetting up and down 10-15 times.
Once the tissue in homogenized, add 9 mL of DMEM 10:10:1DMEM,Gibco - Thermo Fisher ScientificCatalog #11965092 supplemented with 10% Horse SerumGibco - Thermo Fisher Scientific , 10% ofFBSGibco - Thermo Fisher ScientificCatalog #A5209501 , 1% ofPenicillin/streptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4458
Centrifuge samples at 200 x g, 21°C, 00:10:00
10m
Discard the supernatant and add DMEM 10:10:1, resuspend then plate single cell suspension in PDL-coated flasks.
Microglia isolation from mixed glia cultures
10-12 days later, check for microglia on top of the confluent mixed glia layer, then shake cells at 0.42 x g, 02:00:00 on an orbital shaker.
2h
Collect supernatant and spin at 200 x g, 21°C, 00:10:00
10m
Count cells with Trypan blueGibco - Thermo Fisher Scientific and plate at 200,000/well in PDL-coated 12-well plates.
Neuron culture
3h 30m
Coat tissue culture plates
2h
Add 1 mL poly-D-lysineMilliporeSigma (1 mg/mL in H2O ) and 1 mL lamininSigma aldrich.com (0.1 mg/mL in H2O) to 28 mL H2O, mix and add to wells.
Incubate at 37 °C for 02:00:00 . Wash 3x with mqH2O, add MEM,Invitrogen and leave in incubator. Alternatively, let dry after H2O washes and store at 4 °C .
Note
It can be used for at least a week after coating.
Dissection and Trituration
Dissect cortices and midbrain from PND0-1 pups in cold HBSSInvitrogen .
Transfer tissue to warm enzyme solution for 00:30:00 in the water bath (37 °C ), mix periodically.
30m
Wash tissue in 3x 5 mL with plating media.
Note
Move tissue from one tube to the next.
Move tissue to new tube with 1.25 mL plating media. Use a flame-polished glass pipette (approximately half the original diameter) to triturate:
- triturate 3-4x, let tissue settle
- remove supernatant (contains cells)
- add 1.25 mL plating media to remaining tissue
- triturate 3-4x, let tissue settle
- add supernatant to existing tube, mix
Count cells using a hemocytometer.
Plate 70,000/well in 12 well plates.
Maintain cells at 37 °C , 5% CO2.
01:00:00 later, change media to hippocampal feeding media (HFM). then place back in incubator.
1h
Atp13a3 siRNA transfection in microglia
2d 2h 20m
Atp13a3 knockdown was performed using ON-TARGETplus SMARTpool siRNA,Horizon DiscoveryCatalog #L-051693-01-0010 delivered in DharmaFECT transfection reagentHorizon DiscoveryCatalog #T-2005-01 following the instructions of the manufacturer.
The Atp13a3 siRNA target sequences are
5’-GAAGAAAUAAGAUGCGAGA-3’,
5’-CUGCCUGAGUGGCGAGUGA-3’,
5’-GAUACUAGUGGCUUAACUUA-3’, and
5’-UAUCAACAAGGGUCAAGAA-3’, and
non-targeting siRNA, 5’-UGGUUUACAUGUCGACUAA-3’, as control.
A working stock of 1 micromolar (µM) was made using delivery media.
In eppendorf 1, Mix OptiMEM, and siRNA (5 nanomolar (nM) , 10 nanomolar (nM) or 100 nanomolar (nM) ) to a total volume of 64 µL
In eppendorf 2, Mix 2 µL DharmaFECT transfection reagentHorizon DiscoveryCatalog #T-2005-01 with Horizon delivery media to a final volume of 186 µL
Mix eppendorf 1 and 3 then incubate for 00:30:00 at Room temperature and then replace media with 250 µL of the mix to each well.
- Incubate at 5% CO2 and37 °C .
After 24:00:00 , treat microglia with LPSSigma aldrich.comCatalog #serotype 0111:B4 (100 ng/mL ) for 24:00:00
- Add Spd (0.3 micromolar (µM) ) for 02:00:00
- Fix in 4 % volume PFA AT 4 °C for 00:20:00 .
2d 2h 20m
For qPCR, resuspend a subset of microglia in a guanidine-thiocyanate containing lysis buffer then snap-freeze in liquid nitrogen and store at -80 °C .
Microglia treatment and establishing co-cultures
4d 5h 40m
Two to three days following plating in 12-well plates, treat microglia with 100 ng/mL LPS,Sigma aldrich.comCatalog #serotype 0111:B4 inDMEM,Invitrogen 10:10:1 for 24:00:00 .
1d
Treat microglia with Spd (0.01 micromolar (µM) and 0.3 micromolar (µM) )
in combination with pharmacological modulators, such as the polyamine transport inhibitor AMXT 1501 (1 micromolar (µM) pretreatment for01:00:00 prior to LPS,Sigma aldrich.comCatalog #serotype 0111:B4 ), or the mitochondrial antioxidant 1 micromolar (µM) MitoTEMPO Sigma aldrich.comCatalog #SML0737 pretreatment for01:00:00 prior to LPS.
Spd andAMXT 1501 treatments are performed in combination with 1 millimolar (mM) of Aminoguanidine to inhibit polyamine oxidases in the sera.
1h
- Following incubation times, fix microglia in 4 % (v/v) PFA for 00:20:00 at 4 °C then process for immunocytochemistry.
- Alternatively, collect cells in a guanidine-thiocyanate containing lysing buffer, snap-freeze in liquid nitrogen and store at -80 °C for qPCR.
- For mitosox detection, cells were treated with mitosox for 35 minutes at 5% CO2 and37 °C , and imaged on LSM880 confocal microscope.
20m
Spin media supernatant at 300 x g, 4°C, 00:10:00 , store at -80 °C for cytokine analysis.
10m
Microglia-neurons co-cultures:
- For establishing the effects of Spd treatment on neuronal cultures, treat microglia with 0.3 micromolar (µM) Spd for 02:00:00
- Shake at 0.42 x g, 02:00:00 on an orbital shaker.
- Resuspend microglia in 50:50 ratio of Neurobasal medium,Invitrogen and DMEM,Invitrogen 10:10:1.
4h
- Add microglia to the neuronal cultures on DIV11 in 1:4 ratio.
- Maintain co-cultures for 72:00:00 at 37 °C , 5% CO2.
- Fix with 4 % (v/v) PFA for 00:10:00 on ice.
3d 0h 10m
Immunocytochemistry
- Wash coverslips with PBS two times using 500 µL/well, 5 min each.
- Incubate coverslips in 1%FBS + 0.1% BSA in PBS-T using 500 µL/well for 1 h.
- Incubate coverslips with Primary Antibody cocktail: Mix primary antibodies in 1%FBS + 0.1% BSA (determine volume based on number of wells): 500 µL/well and leave overnight at 4°C.
- wash coverslips with PBS two times -5 min each.
-Incubate coverslips in 10% FBS + 1% BSA in PBS-T using 500 µL/well for 15 min at room temperature.
-Incubate with secondary antibodies cocktail and Phalloidin 594 (Thermo Fisheer A12381, lot 261933) in 1%FBS + 0.1% BSA in PBS-T (determine volume based on number of wells) using 300 µL/well for 1.5 h at room temperature.
- Incubate with DAPI (5ug/ml) in 1%FBS + 0.1% BSA in PBS-T (determine volume based on number of wells) using 500 µL/well for 20 min.
- Wash with PBS three times, 5 min each.
-Invert coverslips on glass slides using mounting media appropriate for the microscope objectives used.
Protocol references
Azfar, 2025 #25