Flow cytometric staining of the inflammatory infiltrate at the peak of EAE. (a) CD4 versus CD11b staining of CNS cells (initially gated using a live leukocyte gate). (b) CD11b+ cells, gated on live leukocyte gate. (c) Expression of Ly6c (monocytes) and Ly6g (neutrophils) within the CD11b+ gate. (d) Expression of CD45 on the Ly6c+ (black) or Ly6c− (grey) cells. (e–l) Ly6c+ monocytes in the CNS express high levels of MHC class II, CD64, intermediate expression of CD11c, the costimulatory molecules CD40, CD80, and CD86, and the two ligands for PD-1, CD274 (PD-L1), and CD273 (PD-L2). In histograms (e–l), grey = isotype control, black = specific antibody
Intracellular cytokine staining of CD4+ T cell population retrieved from the CNS at the peak of clinical EAE. CNS mononuclear cells were stimulated overnight +/− antigen prior to staining. (a) Isotype control staining. (b) Unstimulated cells. (c) MOG(35–55)-stimulated cells. Plots are gated on CD4+CD11b− cells
A major problem which challenges studies on monocytes/macrophages and their role in CNS inflammation is the ability to distinguish recruited cells from resident microglia in the CNS as both cell types have similar surface expression of the commonly used markers CD11b and CD45. Ly6c is highly expressed on infiltrating CCR2+ monocytes required for disease progression (2) and more recently has been used to define infiltrating monocytes from resident microglia which are Ly6c− (3, 4). Using the gating strategy outlined here in Fig. 1, retrieved cells are gated on live leukocytes and the CD11b+ cells identified (Fig. 1b). These CD11b+ cells can be subdivided into Ly6g+ (clone 1A8) neutrophils, Ly6c+ infiltrating monocytes, and Ly6c− microglia (Fig. 1c). Both Ly6c+ and Ly6c− populations show similar CD45 expression profiles (Fig. 1d). Having defined the infiltrating monocytes as CD11b+ CD45+ Ly6g− Ly6c+ cells, it is possible to look for further immune activation markers based on expression of MHC class II (Fig. 1e) and costimulatory molecules CD80, CD86, and CD40 (Fig. 1f–h). There has been much debate as to whether the infiltrating monocytes are able to differentiate into either macrophages or dendritic cells at the site of inflammation. In our hands, infiltrating monocytes have high expression of CD64 (Fig. 1I), which is selectively expressed on macrophages rather than dendritic cells (5) and show intermediate expression of CD11c (Fig. 1j) (6). This fits with the ability of monocytes to upregulate CD11c expression at sites of inflammation (7, 8). These Ly6c+ infiltrating monocytes have high expression of both CD273 (PD-L2) and CD274 (PD-L1) important for T cell regulation during inflammation (Fig. 1k–l) (9).
3.2.1 Ex Vivo Surface Staining of Isolated Cells for Flow Cytometric Analysis
Transfer samples to FACS tubes and pellet the cells by centrifugation at 300 × g for 5 min. Discard the supernatant.
Add 2 ml PBS to the cell pellet and centrifuge at 300 × g for 5 min at 4 °C. Repeat this wash step to remove free protein.
Add 2 ml FACS buffer and centrifuge the cells at 300 × g for 5 min at 4 °C, and repeat.
Resuspend the cell pellet in 50 μl FACS buffer containing fluorescently conjugated antibodies for cell surface markers of interest and incubate at 4 °C for 30 min.
Repeat previous wash step 4.
Resuspend cells in 500 μl 1 % PFA or FACS buffer and store at 4 °C prior to acquisition.
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3.2.2 Ex Vivo Transcription Factor Staining of Mononuclear Cells for Flow Cytometry
Once cells are stained for viability and surface molecules (Section 3.2.1), it is necessary to fix and permeabilize the cells prior to staining for transcription factors such as T-bet, RORγt, or Foxp3.
Add 500 μl eBioscience fixation buffer overnight at 4 °C.
Add 2 ml FACS buffer to each tube and centrifuge at 300 × g for 5 min at 4 °C. Discard the supernatant.
Add 50–100 μl 1× eBioscience permeabilization buffer containing relevant antibodies for 30 min at room temperature.
Wash and pellet cells twice in 2 ml FACS buffer.
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