Anti cd11c percp cyanine5

Single-cell suspensions of NALT cells were stained for 30 min at 4°C with appropriate combinations of fluorochrome-conjugated maternal antibodies in the presence of 0.2% bovine serum albumin (BSA) and fixed in 4% paraformaldehyde in PBS. Cells were stained for surface markers with anti-CD284 (TLR4)-PE-Cyanine7 (SA15-21, Biolegend) or anti-CD284 (TLR4)-PE (SA15-21, Biolegend), anti-CD11c-PE (N418, eBioscience), or anti-CD11c-PerCP-Cyanine5.5 (N418, eBioscience), anti-CD19-APC (6D5, Biolegend), anti-CD3-FITC (17A2, eBioscience), anti-CD11b-PE-Cyanine7 (M1/70, eBioscience), anti-Ly-6G/Ly-6C (Gr-1)-APC (RB6-8C5, Biolegend), anti-F4/80-PE (BM8, eBioscience), and anti-CD282 (TLR2)-PE (CB225, Biolegend) as needed. For intracellular staining, fixed cells were permeabilized and stained in saponin (0.1% in PBS; Sigma) with anti-CD289 (TLR9)-FITC (M9.D6, eBioscience) and anti-CD283 (TLR3)-PE (11F8, Biolegend) in the presence of 1.5% BSA. Samples were analyzed using a FACSCanto flow cytometer (BD Biosciences) and FlowJo software (Treestar, Ashland, OR).

Six days post-infection, mice were exsanguinated and perfused as above; brains were harvested and homogenized in RPMI media-containing 0.5 mg/mL collagenase (Gibco LifeTechnologies), 0.01 mg/mL DNAse I (Roche) and 2 mM EDTA. Infiltrating cells were separated on a 33.3 % Percoll solution. Cells were stained for flow cytometry analyses with the following; Zombie Aqua Dye-V500 (BioLegend), anti-CD45-APC-eFluor780 (clone 30-F11, eBioscience), anti-CD4-PerCP Cyanine5.5 (clone RM4-5, eBioscience), anti-CD8alpha-PE (clone 53-6.7, eBioscience), anti-TCRbeta-FITC (clone H57-597, eBioscience), anti-CD19-BV421 (clone 6D5, BioLegend), F4/80-eFluor450 (clone BM8, eBioscience), anti-CD11b-APC (clone M1/70, eBioscience), anti-CD11c-PerCP Cyanine5.5 (clone N418, eBioscience), anti-Ly6C-PE (clone HK1.4, eBioscience), and anti-Ly6G-FITC (clone 1A8, BioLegend). Leukocytes were gated as CD45^hi cells.