Pe cy5 cd21 b ly4

Previously frozen PBMCs were incubated in 1 mL of pre-warmed complete media (RPMI 1640 supplemented with 10% FBS and 2mM L-glutamine) containing 20 nM of MitoTracker Green (Invitrogen) at 37°C for 30 mins. Cells were collected by centrifugation then resuspended in 10 μL of warm complete medium and incubated at 37°C for 30 mins. Next, the cells were stained on ice for 30 mins in 100 μLs FACS buffer (PBS + 0.5% BSA) containing 5% normal mouse serum, 5% normal rat serum and the following fluorochrome-conjugated mouse anti-human monoclonal antibodies: PE-IgD (IA6-2, BD Biosciences), PE-A610-CD24 (SN3, Invitrogen), PE-Cy5-CD21 (B-ly4, BD), PerCP-Cy5.5-CD38 (HIT2, BD), PE-Cy7-CD23 (EBVCS2, eBioscience), Pacific Blue-CD3 (SP34-2, BD), Qdot605-CD27 (CLB-27/1, Invitrogen), allophycocyanin-CD95 (DX2, BD), allophycocyanin-Cy7-CD19 (SJ25C1, BD Biosciences), as well as the biotinylated 9G4 rat anti-human Ig idiotype antibody, which was detected by a further staining with 100 μL of streptavidin-Alexa680 (Invitrogen) at a 1:500 dilution in FACS buffer on ice for 30 mins. Cells were washed with 3 mLs PBS and stained with 1 mL LIVE/DEAD aqua-fluorescent reactive dye (Invitrogen) at a 1:1000 dilution in PBS on ice for 30 mins. After resuspension in FACS buffer, cells were acquired on a LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).