Cd38 apc hb 7

EDTA anticoagulated peripheral blood cells were labeled following a lyse/wash protocol with an 8-color/9-monoclonal antibody (mAb) panel including CD3 AmCyam (clone SK7, BD Biosciences), CD4 PECy7 (SK3, BD), CD8 APCCy7 (SK1, BD), CD16 PacBlue (3G8, BD), CD19 PECy7 (SJ25C1, BD), CD28 FICT (CD28.2, BD), CD38 APC (HB7, BD), CD86 PE (IT2.2, BD), and HLA-DR PerCP (L243, BD). Five microliters of each antibody in 100 µL of whole blood was incubated for 15 minutes at room temperature in the dark. Samples were lysed with 3 mL of 1X FACSlysing solution (BD) for 5 minutes and washed with 3 mL of FACSFlow (BD). Half a million cells were immediately acquired in a FACSCanto flow cytometer (BD), daily calibrated using 7-color setup beads (BD), and analyzed with DIVA software (BD) following the gating strategy described in Supplementary Figure 1.
The expression of CD28, CD38, CD86, and HLA-DR activation/senescence markers was evaluated as a percentage or absolute number (cells/µL) of positive cells as well as mean fluorescence intensity (MFI) of the marker on CD3⁺CD4⁺, CD3⁺CD8⁺, and CD3⁺CD4⁺CD8⁺ T lymphocytes, CD19⁺ B lymphocytes, CD3^-CD19^-CD16⁺ natural killer (NK) lymphocytes, monocytes (CD4⁺CD86⁺HLA-DR⁺ medium side scatter [SSC] cells), granulocytes (CD16⁺⁺ elevated SSC cells), and eosinophils (elevated SSC auto fluorescent cells).