Anti cd44 percp

Mice were sacrificed and spleens were harvested at 24h after CLP. Splenocytes were stained with anti-CD3-Alexa 700 (BD), anti-CD4-PB (Biolegend, clone RM4–5), anti-CD8-PO (Invitrogen, clone MCD0830), anti-CD44-PerCP (Biolegend, clone IM7), anti-CD62L-PE (BD), anti-CD28-PE-Cy7 (Biolegend, clone E18) and anti-CD25-APC-Cy7 (BD). For detection of cell apoptosis, splenocytes were stained with a FITC Annexin V apoptosis detection kit with 7-AAD (Biolegend). Anti-Bcl-xL (54H6) and Bcl-2 (Biolegend, clone BCL/10C4) were used to detect engagement of the mitochondrial pathway of apoptosis while anti-CD95 (Biolegend, clone DX2) and anti-TNFR Type Ⅰ (Biolegend, clone 55R-286) were stained to detect expression of death receptors on T cells. Cells were intracellularly stained with anti-Ki-67 (Biolegend, clone 16A8) to assay for cell proliferation. Tregs were identified via intracellular staining for Foxp3-FITC (Ebioscience, clone FJK-16S) using the Foxp3 staining kit (Ebioscience). B cells were stained with anti-CD19-FITC (Biolegend). NK cells were stained with anti-NK1.1-PE (Ebioscience). Dendritic cells were stained with anti-CD11c-PE-Cy7 (BD). Neutrophils were stained with anti-Gr-1-Alexa 700 (Biolegend) and anti-CD11b-PerCP (Biolegend). Accucheck Counting Beads (Thermo Fisher Scientific) were added after staining to calculate the absolute number of cells per spleen.