Application: |
Primary cell isolation |
Cells used: |
PBMC from buffy coat |
Brief Protocol: |
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. |
Results Summary: |
Very good purity achieved with both antibody dilutions, so 1:4 dilution recommended. |
Additional Notes: |
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-Cyanine7. CD4 ab: SK3-APC-Cyanine7. |
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