Fvd efluor450

At 72 hours after GMH, under deep isoflurane anesthesia, blood samples were collected via cardiac puncture and mixed with citrate-phosphate dextrose anticoagulant solution (Sigma-Aldrich) in a 1:5 ratio. Peripheral blood mononuclear cells (PBMCs) were then isolated by ammonium chloride lysis of red blood cells. PBMCs were then washed and pelleted by centrifugation (1500 rpm for 5 minutes at 4 °C). The resulting cell suspensions were counted and diluted to approximately 1×106 cells/ml. For multi-color flow cytometry staining, cells were incubated with monoclonal antibodies in PBS for 30 minutes in the dark at 4 °C. Cells were subsequently washed and fixed with 1% paraformaldehyde/PBS before analysis on a MACSQuant Analyzer (Miltenyi Biotec). The anti-rat antibodies used were CD45-APC-eFluor780, CD3-APC (clones: Ox1 and eBioG4.18 respectively, eBioscience, San Diego, CA) and CD11b-PE (clone: Ox42, Biolegend, San Diego, CA). Isotype controls consisted of mouse IgG1κ-APC-eFluor780, IgG1κ-APC and IgG2a,κ-PE. Living cells were identified by forward scatter and side scatter gating along with exclusion of the fixable viability dye FVD eFluor450 (eBioscience), added immediately prior to fixation. Flow cytometry data analysis was conducted using Flowjo data analysis software (Tree Star, Ashland, OR).