Pe conjugated anti cd38

Control Sirt3^+/+ and Sirt3^−/− mice were immunized intraperitoneally at 8 to 12 weeks of age with 0.5 ml suspension of 2% sheep red blood cell (SRBC) in PBS (Cocalico Biologicals) to induce GC formation. Mice were sacrificed after 10 days and spleens were isolated from control and SIRT3 knockout mice. The spleen sections derived from Sirt3^+/+ and Sirt3^−/−mice were stained by hematoxylin and eosin (FI&E) and peanut agglutinin (PNA) using standard procedures. The number of GCs, the total spleen area occupied by GCs and the average area occupied by the GCs were quantified using ImageJ 1,44o (NIH) software. To determine the percentage of GC B-cell population, single-cell suspensions from spleens derived from Sirt3^+/+ and Sirt3^−/− mice were stained using the following fluorescent-labeled anti-mouse antibodies: FITC conjugated anti-B220, PE conjugated anti-FAS, APC conjugated anti-CD38 from BD biosciences and analyzed by flow-cytometry. To evaluate follicular and marginal zone B-cell populations, splenic B cells were stained with APC conjugated anti-B220, FITC conjugated anti-CD21 and PE conjugated anti-CD38 from BD biosciences and then analyzed by flow-cytometry. DAPI was used for the exclusion of dead cells. The data was analyzed by FlowJo software.

T cells were quantified by size, granularity and positive expression of CD3 and either CD4 or CD8. T-cell activation was determined using PE-conjugated anti-CD38, FITC-conjugated anti-HLA-DR, peridinin-chlorophyll-protein-complex-conjugated anti-CD3, allophycocyanin-cy7-conjugated anti-CD8 and allophycocyanin-conjugated anti-CD4 antibodies all from BD Biosciences. T-cell activation was defined as expression of co-expression of CD38 and HLA-DR. Measurements were performed using a LSR II flow cytometer and analyzed using FACSDiva software.