Analysis of Immune Cells in Mouse Nervous Tissues Using Flow Cytometry

Abstract

Source: Hidmark, A. S., et al. Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry. J. Vis. Exp. (2017).

This video demonstrates the use of flow cytometry to analyze immune cells in mouse sciatic nerves and dorsal root ganglia (DRGs). The process involves labeling immune cells with fluorophore- and biotin-tagged antibodies, stabilizing and permeabilizing the cells, and using fluorescent markers to detect cell-surface and nuclear characteristics. The technique enables a quantitative assessment of immune cell heterogeneity in nervous tissues.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6 Mouse	Charles River	C57BL/6NCrl
DMEM (+1g/L glucose, Glutamine, Pyruvate)	Thermo	31885023	The source of this material is not important
HEPES	Sigma-Aldrich	H3375
Bovine serum albumin (BSA)	Sigma-Aldrich	A2153
Collagenase Type 4	Worthington Biochemical Corp., US	LS004188
Deoxyribonuclease (DNase) I from bovine pancreas I	Sigma-Aldrich	DN25-1g
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	E6758
Foetal Calf Serum (FCS), heat inactivated	Sigma-Aldrich	F4135
Antibody against CD11b-PerCP/Cy5.5	Biolegend	101228	Clone M1/70
Antibody against MHC class II, biotinylated	Biolegend	107603	Clone M5/114.15.2
Antibody against CD45-A647	Biolegend	107603	Clone 30-F11
Streptavidin-PE/Cy7	Biolegend	405206	Used to detect MHCII-biotin with CD45-A647 and CDllb-PerCP/Cy5.5
Streptavidin-PerCP	Biolegend	405213	Used to detect MHCII-biotin with CD68-APC and F4/80-PE/Cy7
Triton-X 100	Sigma-Aldrich	T8787
DAPI	Sigma-Aldrich	D9542-1MG	Dilute in PBS to a 50x and store at 4 °C in the dark
Cell dissociation sieve - tissue grinder kit	Sigma-Aldrich	CD1
V-Shaped, 96-well plates	Greiner/Sigma	M8185
5ml polystryene round-bottom tube, 12x75mm	BD Biosciences	352008
60x15mm petri dish	Greiner/Sigma	Z643084
BD LSR II Flow Cytometer	BD Biosciences