Human apheresis samples were obtained from unidentified healthy human donors. To isolate PBMCs, blood was layered onto Lymphoprep (PAA) and isolated by density-gradient centrifugation. Interface cells were washed twice in PBS, then once in MACS buffer before undergoing magnetic bead selection with Miltenyi CD4 Negative selection kit II, CD8 Negative Selection Kit, CD4 Memory T cell selection kit, or the B Cell Isolation Kit II according to manufacturer instructions. For effector cell isolation, CD4 T cells isolated with CD4 Negative selection kit II were subsequently depleted of CD27+ cell types with Miltenyi CD27 positive selection beads.
Where indicated, before culture, T or B cells were labeled with 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) as previously described [18 (link)]. Briefly, labeling was performed by incubating cells at 106 cells/ml at 37°C for 10 minutes with 5uM CFSE in PBS containing 0.1% bovine serum albumin (BSA). CFSE was quenched by adding twice volume of complete media, followed by 3 washes in complete media.
Where indicated, before culture, T or B cells were labeled with 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) as previously described [18 (link)]. Briefly, labeling was performed by incubating cells at 106 cells/ml at 37°C for 10 minutes with 5uM CFSE in PBS containing 0.1% bovine serum albumin (BSA). CFSE was quenched by adding twice volume of complete media, followed by 3 washes in complete media.