TITLE: Microglia validation by FACS-analysis
AUTHORS: Riana Lo Bu, Frank Soldner

[DESCRIPTION]
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.

[STEPS]
SECTION: Microglial precursor validation
1. Harvest microglial precursors from flasks in STEP4 by collecting the media into 15 ml conical tubes and centrifuging it at150 rcf for 4 min.   

SECTION: Microglial precursor validation
2. Remove the supernatant and resuspend the cells in STEP4 media.
SECTION: Microglial precursor validation
3. Count the cells and aliquot 100,000 cells/experiment condition in 15ml conical tubes.
SECTION: Microglial precursor validation
4. Spin down the cells (150 rcf for 4 min).

SECTION: Microglial precursor validation
5. Remove the supernatant and resuspend the cells in 100 µL FACS buffer (PBS1x, EDTA 5 mM, 0.5% FBS).

SECTION: Microglial precursor validation
6. Add 5 µL/tube of human TruStain FcX and incubate for 10 min at Room temperature.

SECTION: Microglial precursor validation
7. Add primary antibodies for the desired experimental conditions (1 µL/tube for the antibodies described above or according to manufacturer's recommendations).

SECTION: Microglial precursor validation
8. Incubate for 30 min on on ice.

SECTION: Microglial precursor validation
9. Wash the cells 2 times by adding 2 mL/tube of FACS buffer followed by centrifugation at 150 rcf for 4 min

SECTION: Microglial precursor validation
10. Resuspend the cells in 350 µL of FACS buffer + calcein violet (follow manufacturer’s instructions as to how to prepare calcein violet).

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

SECTION: Mature Microglia validation
13. Incubate 10 min at 37 °C.

SECTION: Mature Microglia validation
14. Add 3 mLof microglia maturation media to quench the reaction and collect the cells in 15 ml conical tubes.

SECTION: Mature Microglia validation
15. Spin down at 150 rcf for 8 min.

SECTION: Mature Microglia validation
16. Remove the supernatant and resuspend the cells in microglia maturation media.
SECTION: Mature Microglia validation
17. Count the cells and aliquot 100,000 cells/experiment condition in 15ml conical tubes.
SECTION: Mature Microglia validation
18. Spin down the cells (150 rcf for 4 min).

SECTION: Mature Microglia validation
19. Remove the supernatant and resuspend the cells in 100 µLFACS buffer (PBS1x, EDTA 5 mM, 0.5% FBS).

SECTION: Mature Microglia validation
20. Add 5 µL/tube of human TruStain FcX and incubate for 10 min at Room temperature.

SECTION: Mature Microglia validation
21. Add primary antibodies for the desired experimental conditions (1 µL/tube for the antibodies described above or according to manufacturer's recommendations).

SECTION: Mature Microglia validation
22. Incubate for 30 min on ice.

SECTION: Mature Microglia validation
23. Wash the cells 2 times by adding with by adding 2 mL/tube of FACS buffer followed by centrifugation at 150 rcf for 4 min.

SECTION: Mature Microglia validation
24. Resuspend the cells in 350 µL of FACS buffer + calcein violet (follow manufacturer’s instructions as to how to prepare calcein violet).

SECTION: Mature Microglia validation
25. Keep your samples protected from light on in ice and perform standard FACS analysis.