Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Summary

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Abstract

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated ≥ 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Introduction

Immune cells which enter the PNS from the circulation, as defined by the expression of CD45 and CD11b, help to maintain the integrity of the nerve and play a role both in regeneration and degeneration¹. Macrophages (defined by their expression of CD68 in mouse) can be skewed towards an inflammatory phenotype, expressing more MHC class II and CD86 on their surface (M1), or towards an anti-inflammatory phenotype, expressing more intracellular CD206 (M2)². Skewing of the macrophage phenotype is a dynamic process regulated through Akt signaling³, reflecting the different tasks of macrophages in the de....

Protocol

Wild-type C57BL/6 mice (Males; 10-12 weeks) were kept in standard 12 h light/dark cycle and were provided free access to standard chow diet and water. All animal experiments were conducted in accordance to the relevant guidelines by the local Animal Care and Use Committee and approved by the local Animal Care and Use Committee at the regional authority in Karlsruhe, Germany (G216/10).

1. Perfusion of the Mouse

1. Sacrifice the mouse (C57BL/6; Males; 10-12weeks) using CO₂ or in accordance with local regulations.
   NOTE: Retro-orbital bleeding can be performed to obtain control leukocytes, using EDTA-coated 1.5 mL tub....

Results

Cell suspensions from both full-length sciatic nerves and all major DRG were prepared, according to the protocol, from six healthy C57BL/6 mice and divided into three equal aliquots for staining. Counter staining with DAPI, which stains DNA, allows for the detection of a single cell population (Figure 1A-B, upper panel). Washing out the DAPI, prior to analysis by flow cytometry, decreased to an extent the fluorescent intensit.......

Discussion

Sciatic nerves contain a large proportion of lipids, such as cholesterol, due to the content of myelin around the axons. Since the properties of lipids change with temperature, different results may be obtained at different temperatures. To ensure cell preservation, all steps in this protocol after the digestion were performed on ice. Whilst consistency is recommended for the sake of reproducibility of the results, it may be possible to increase yield by performing the steps 3.6 and 3.7 at room temperature. If the nerves.......

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
Antibody against CD68-APC	Biolegend	137007	Clone FA/11
Antibody against CD206-PE	Biolegend	141706	Clone C068C2
Antibody against F4/80-PE/Cy7	Biolegend	123113	Clone BM8
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