||Primary cell isolation
||PBMC from buffy coat
||PBMCs were isolated from buffy coats by Ficoll density centrifugation and cultured in IL-2 media overnight.
CD4 T cells were isolated following the manufacturer's protocol: Negative Selection Protocol for the use MojoSort™ Reagents in Columns.
Two antibody dilutions were tested: 1:2 with 1:5 dilution of SA-beads, and 1:4 with 1:10 dilution of SA-beads. Columns used were MACS LS columns.
After the last wash cells were stained with CD3 and CD4 Ab (along with a pre-sort sample) and acquired on BD LSRII.
||Very good purity achieved with both antibody dilutions, so 1:4 dilution recommended.
||Lymphocytes were gated on FCS vs SSC plot. Doublets were excluded on FSC -H vs -W plot. Dead cells were excluded as Zombie UV+. CD3 Ab: UCHT1-PE-Cy7. CD4 ab: SK3-APC-Cy7.