Anti cd8 pacificblue dk25

For cell death analysis PBMCs were washed in FACS buffer. Fc-receptors were blocked with anti-IgG (Jackson IR). Cells were incubated for 15 min at RT in binding buffer containing anti-CD4-APC (RPA-T4, eBioscience) and anti-CD8-PacificBlue (DK25, Dako) antibodies for cell surface staining as well as FITC Annexin V (BD Pharmingen). To discriminate between viable cells, cells that were in early apoptosis and cells that were in late apoptosis or already dead 1 mg/ml of the vital dye PI (Sigma Aldrich) was added 5 min prior to cell acquisition by flow cytometry. To investigate the influence of edelfosine on MHC class II expression by human B and T cells, cells were stained for viability by using the LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen). Cells were washed with PBS, blocked and stained for cell surface antigen expression as described before. Antibodies were anti-CD3-PE-Cy7 (UCHT1), anti-CD4-APC (RPA-T4), both from eBioscience, anti-CD8-PacificBlue (DK25, Dako), anti-CD5-PerCP-Cy5.5 (L17F12), anti-CD19-V450 (HIB19), anti-IgD-PE (IA6-2) and anti-HLA-DR/DP/DQ-FITC (Tu39), all from BD Biosciences, as well as anti-CD27-APC-Alexa750 (CLB-27/1) and anti-CD45RA-PE-Cy5.5 (MEM-56), both from Invitrogen. Data was acquired on an LSRII flow cytometer (BD Biosciences) and analyzed with FACSDiva (BD Biosciences) and FlowJo (Tree Star) software.