Spleens were removed from mice and single-cell suspensions were prepared by mashing the spleen using a 3-ml syringe plunger on a strainer (70 μM) and washing cells with PBS. Single cell suspensions were stained for flow cytometric analysis with anti-CD3-APC, anti-CD4-Pac Blue, anti-CD8-Pac Orange, Anti- B220-Per CP, anti-annexin-FITC, and anti PI-PE (BD Pharmingen, San Diego, CA). Blood and peritoneal fluid were also harvested and single-cell suspensions were prepared. Cells were stained with Gr-1-FITC, B220-Per CP, CD11b-APC, CD3-Pac Blue, CD11c-PE-Cy7; or CD4-Pac Blue, CD8-Pac Orange, Ly49D-FITC, NK1.1-PE, CD127-APC.
To measure production of cytokines on a per cell basis, splenocytes were stimulated with phorbol 12-myristate 13-acetate (PMA, 30 ng/mL) and ionomycin (400 ng/mL) in the presence of 10 μg/mL of Brefeldin A. After 18 hours, cells were surface stained with anti-CD4 and anti-CD8 and processed with an intracellular staining kit (BD Biosciences) according to manufacturer’s instructions. Intracellular antibodies included anti-interferon (IFN)-γ (eBioscience, San Diego, CA), anti-TNF, and anti-IL-2 (both BD Biosciences). Data were acquired on a LSR II multicolor flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar, San Carlos, CA).
To measure production of cytokines on a per cell basis, splenocytes were stimulated with phorbol 12-myristate 13-acetate (PMA, 30 ng/mL) and ionomycin (400 ng/mL) in the presence of 10 μg/mL of Brefeldin A. After 18 hours, cells were surface stained with anti-CD4 and anti-CD8 and processed with an intracellular staining kit (BD Biosciences) according to manufacturer’s instructions. Intracellular antibodies included anti-interferon (IFN)-γ (eBioscience, San Diego, CA), anti-TNF, and anti-IL-2 (both BD Biosciences). Data were acquired on a LSR II multicolor flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar, San Carlos, CA).