Anti 2b4 apc

2x10⁶ splenocytes from each sample were plated in a 96-well plate. After centrifugation, cells were resuspended and incubated in culture medium (R10) consisting of RPMI 1640 containing 10% FBS (Mediatech, Herndon, VA), 2mM L-glutamine, 0.01 M HEPES buffer, 100mg/ml gentamicin (Mediatech), and 5×10^-5M 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). To test intracellular cytokine, cells were stimulated with 30 mg/ml PMA and 400 ng/ml ionomycin in the presence of GolgiStop (BD Pharmingen) for 4 hours at 37°C.
After incubation and stimulation, cells were surface-stained with anti-CD3-PB (BD), anti-CD4-PerCP (BD), anti-CD8-PO (Biolegend), anti-BTLA-PE, anti-2B4-APC, anti-PD-1-APC-Cy7 (all from eBioscience). Then cells were permeabilized using fixation and permeabilization solution (BD). We used anti-IL-2-FITC (BD), anti-TNF-PE-Cy7 (Biolegend) and anti-IFN-γ-Alexa 700 (BD) for intracellular cytokine staining. In some experiments, cells were stained intracellularly for Ki-67 using the Foxp3 staining kit (BD Pharmingen). Samples were analyzed on an LSRII flow cytometer (BD) and data were analyzed using FlowJo software (FlowJo, LLC) and SPADE (Cytobank.org) (see below).

Mice were sacrificed and spleens were harvested 24-hours post CLP. Splenocytes were stained with anti-CD3-Alexa 700 (BD), anti-CD4-PB (BD), anti-CD8-PO and anti-CD44-PerCP (Biolegend). Cells were also surface stained with anti-CD25-FITC (Biolegend), anti-CD69-PE (Biolegend), anti-CD62L-PE Cy7 (BD), and anti-BTLA-PE, anti-2B4-APC, anti-PD-1-APC-Cy7, anti-LAG-3-FITC (all from eBioscience) for phenotypic analysis. An LSR II flow cytometer (BD Biosciences) was used to run all samples. Accucheck Counting Beads (Thermo Fisher Scientific) were added during staining to calculate the absolute number of T cells per spleen. Flow data were analyzed using FlowJo software (FlowJo, LLC) and SPADE (Cytobank.org) (see below).