Sep 29, 2025

Microglia validation by FACS-analysis

DOI
https://dx.doi.org/10.17504/protocols.io.81wgbxokqlpk/v1
  • 1Albert Einstein College of Medicine, 1301 Morris Park Ave., Bronx, NY 10461, USA.;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815.
Oriol Busquets
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DOI: https://dx.doi.org/10.17504/protocols.io.81wgbxokqlpk/v1
Protocol Citation: Riana Lo Bu, Frank Soldner 2025. Microglia validation by FACS-analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxokqlpk/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: January 08, 2024
Last Modified: September 29, 2025
Protocol  Integer ID: 93109
Keywords: ASAPCRN, microglia validation by fac, microglia validation, microglia validation by fluorescence, microglia differentiation protocol, procedure for microglial precursor, microglia differentiation protocol for cell generation, microglial precursor, microglia differentiation, activated cell sorting, cell sorting, fac, list of reagent
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000486
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-024409
Abstract
This protocol describes the procedure for microglial precursor and mature microglia validation by Fluorescence-Activated Cell Sorting (FACS) analysis. Please refer to (https://doi.org/10.17504/protocols.io.4r3l22zbjl1y/v1) for microglia differentiation protocol for cell generation and media composition.

Protocol Overview

A. Microglial precursor validation
B. Mature microglia validation

General Notes

A list of reagents and relevant vendor information can be found in the table listed under the materials tab.
Attachments
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Microglia validation...
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Materials
Reagent Table:
ItemVendorCatalog Number
1xPBSCorningMT21031CV
AccutaseGemini400-158
EDTAFisherBP120500
Trypan blueGibco15250061
Human TruStain FcXBiolegend422302
APC Anti-human CD16Biolegend302102
APC Anti-human CD45Biolegend304012
APC anti-human CX3CR1Biolegend341610
PE Anti-human P2ry12Biolegend392103
PE Anti-human CD11bBiolegend301306
PE Anti-human CD14Fisher12014942
Calcein violetInvitrogenC34858

Microglial precursor validation
52m
Harvest microglial precursors from flasks in STEP4 by collecting the media into 15 ml conical tubes and centrifuging it at150 rcf for 00:04:00 .

4m
Remove the supernatant and resuspend the cells in STEP4 media.
Count the cells and aliquot 100,000 cells/experiment condition in 15ml conical tubes.
Spin down the cells (150 rcf for 00:04:00 ).

4m
Remove the supernatant and resuspend the cells in 100 µL FACS buffer (PBS1x, EDTA 5 mM, 0.5% FBS).

Add 5 µL /tube of human TruStain FcX and incubate for 00:10:00 at Room temperature .

10m
Add primary antibodies for the desired experimental conditions (1 µL /tube for the antibodies described above or according to manufacturer's recommendations).

Incubate for 00:30:00 on On ice .

30m
Wash the cells 2 times by adding 2 mL /tube of FACS buffer followed by centrifugation at 150 rcf for 00:04:00

4m
Resuspend the cells in 350 µL of FACS buffer + calcein violet (follow manufacturer’s instructions as to how to prepare calcein violet).

Keep your samples protected from light on ice and perform standart FACS analysis.
Mature Microglia validation
32m
Remove the media from microglia cultures and add accutase (1 mL /well in 6 well plates).

Incubate 00:10:00 at 37 °C .

10m
Add 3 mL of microglia maturation media to quench the reaction and collect the cells in 15 ml conical tubes.

Spin down at 150 rcf for 00:08:00 .

8m
Remove the supernatant and resuspend the cells in microglia maturation media.
Count the cells and aliquot 100,000 cells/experiment condition in 15ml conical tubes.
Spin down the cells (150 rcf for 00:04:00 ).

4m
Remove the supernatant and resuspend the cells in 100 µL FACS buffer (PBS1x, EDTA 5 mM, 0.5% FBS).

Add 5 µL /tube of human TruStain FcX and incubate for 00:10:00 at Room temperature .

10m
Add primary antibodies for the desired experimental conditions (1 µL /tube for the antibodies described above or according to manufacturer's recommendations).

Incubate for 00:30:00 On ice .

30m
Wash the cells 2 times by adding with by adding 2 mL /tube of FACS buffer followed by centrifugation at 150 rcf for 00:04:00 .

4m
Resuspend the cells in 350 µL of FACS buffer + calcein violet (follow manufacturer’s instructions as to how to prepare calcein violet).

Keep your samples protected from light on in ice and perform standard FACS analysis.
Protocol references