May 23, 2022

Public workspaceMicroglia FACS staining after isolation (from UCSD)

DOI
dx.doi.org/10.17504/protocols.io.dm6gpbxp8lzp/v1
  • 1California Institute of Technology
avatar.png
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.dm6gpbxp8lzp/v1
Protocol Citation: rabdelha 2022. Microglia FACS staining after isolation (from UCSD). protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpbxp8lzp/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: May 17, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 62713
Keywords: Microglia FACS staining, antibodies (CD11b, CD45), ASAPCRN
Abstract
This protocol details about microglia FACS staining after isolation (from UCSD).
Attachments
Icon for 437-925.docx
437-925.docx
101KB
Guidelines
Gating Notes
  • live vs dead
  • FSC vs SSC
  • singles vs. doublets.
  • CD11B (high) vs. CD45 (low).
  • CX3CR1 (optional) gate on positive cells.

Misc. Notes
  • **don’t need to compensate with only 2-3 colors.
  • CD45, CD11B, and CX3CR1 are all surface markers.
  • Usually get 20-50% live cells after staining.
  • Better to do staining right after microglia prep rather than waiting O/N for staining.

Processing Notes
Can do RNA seq or Atac Seq
RNA Seq
  • Spin down sample in Eppendorf (not in facs tubes
  • Remove supernatant
  • Add Amount150 µL of trizol → resuspend
  • Store in Temperature-80 °C (can keep stored for months).
ATAC Seq
  • Do transposase reaction and then freeze for processing.

Materials
Materials
use 5ml Polyproprylene eppendorf tubes

Antibodies

  • ReagentCD11b Monoclonal Antibody (M1/70) APCeBioscience/InvitrogenCatalog #17-0112-82
  • ReagentAlexa Fluor® 488 anti-mouse CD45 AntibodyBioLegendCatalog #103122
  • ReagentCD16/CD32 Monoclonal Antibody (93)eBioscience/InvitrogenCatalog #14-0161-82
  • DAPI 1:10,000, for 45 sec, wash twice with PBS; dilute in H2O



Protocol
Protocol
25m 45s
25m 45s
Resuspend cells staining buffer (Amount300 µL of HBSS+EDTA+BSA).
  • Resuspend in a 15-ml falcon tube.
Add Fc Block (~1:100 dilution) and incubate for Duration00:15:00 at Temperature4 °C or in the fridge.
  • Add this to cells resuspended in the HBSS.
15m
Incubation
Pipetting
Take ~5% of cells for unstained control, put TemperatureOn ice .
Note
Already did this during the isolation during the percoll separation step.


Add antibodies (CD11b, CD45, 1:100 dilution) for 20-30 min at Temperature4 °C .
Pipetting
Mix
Add directly to cells in the FC block.
Pipetting
Mix by tapping tube; do not vortex.
Note
Don’t add to unstained cells!!

Mix
Add DAPI, Duration00:00:45 , then dilute with HBSS+BSA+EDTA.
45s
Pipetting
Add 1:1000 dilution.
Add to unstained cells.
Put 70-μm filter on top of a FACS test tube and filter resuspended pellet into the tube.
Note
Do this for unstained samples as well.


Push filter hard onto tube.
Centrifuge for Centrifigation400 x g, 00:10:00 .
10m
Centrifigation
Prepare collection tubes during spin time.
  • Coat FACS tubes with Amount1 mL of staining buffer to prevent cells from sticking to walls of tubes.
  • Invert tube several times.
Vacuum out supernatant.
  • Remove most of supernatant; not all.


Resuspend pellet in Amount500 µL to Amount1000 µL staining buffer.
Note
Depending on machine.

Keep cells on ice the whole time.