Shreffler:CFSE Labeling

{Shreffler}

CSFE Labeling

Suspend PBMCs at 10x106 cells/mL in PBS alone. Cells must be in PBS for CFSE labeling. If CFSE negative control is needed, remove cells now. Create solution of PBS:5μM CFSE CSFE is found in -20 degree fridge in 17-46. There are 2 CFSE stocks available - 40mM and 5mM. Want to start with 10μ M (“2X”). Add 4mL PBS + 8 μL 5 mM CFSE (for example) Combine 1:1 PBMC cells + CFSE (i.e. 300uL PBMCs + 300uL CFSE) in 15 ml tube. Place in H2O bath x 10 min. Wash in 10 mL AIM-V (10 min at 300G). Aspirate. If plating... resuspend in AIM-V to make 4x106 cells/mL (2X). There are now 2 million cells/ 0.5 mL. Cells in tube are now all CFSE labeled and can be plated. If CFSE positive control is needed, remove cells now. If conducting Robocep selection, resuspend in Robocep buffer at appropriate concentration and follow protocol.