Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Summary

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

Abstract

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

Protocol

Preparation of reagents

Prepare stock isotonic percoll (SIP), 4 ml per brain, by mixing 9 parts of percoll with one part of 10X HBSS without Ca++ and Mg++.

Prepare SIP at 70% in 1X HBSS without Ca++ and Mg++, 2 ml per brain dispensed into a 15 ml polypropylene conical tube.

Note: It is recommended that percoll should be used at room temperature, if used cold, cells tend to clump and cell separation is less efficient.

Tissue collection and homogenization

Anesthetize mice and perfuse through the left ventricle with ice-cold 1X HBSS....

Discussion

Analyses of cell surface markers in CNS leukocytes from normal and inflamed mouse tissues has been utilized for several decades^1-3. Isolation protocols are based in the separation of microglia and leukocytes by density centrifugation⁴. Here we describe a fast and effective method of isolating CNS leukocytes using discontinuous percoll gradients. After isolation of cells, staining with various antibodies such as -but not limited to- CD45, CD4, CD8, CD11b, CD19, etc. allows the identification of immun.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tissue homogenization and Gradients Percoll (Amersham) 10X Hanks’ Balanced Salt Solution, without Calcium and Magnesium (Gibco) RPMI Medium 1640, without phenol red (Gibco) 7-ml or 15-ml Dounce Tissue Grinders (VWR) Flow cytometry Cell Staining Buffer (Biolegend) Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block), BD Pharmingen Hemacytometer (Fisher) Anti-mouse CD45 (clone 30-F11), CD4 (clone RM4-5), CD19 (clone 6D5), BioLegend 10X Phosphate Buffered Saline, without Calcium and Magnesium (Thermo Scientific) Paraformaldehyde (Sigma Aldrich) Flow cytometry analysis performed with a LSR II (BD Biosciences)