Buffy Coat PBMC - Single Cell Suspension
Dear Cytoforum,
Typically, my group rests cell samples over night (O/N) at 37*C in 2mM EDTA/10% Serum/PenStrep/RPMI 1640 cell culture medium before analyzing phospho-proteins by CyTOF. Our Buffy Coat PBMC samples tend be highly sticky, as compared to normal PBMC. Even after a brief 25U/mL benzonase treatment, and strain through a 40um filter, the cells rested O/N will clump to the point that they can not be separated by pipetting.
I was curious if anyone has a good protocol for single-cell suspension of resting cells that might be a little more rigorous than what I have been doing, that works for phospho-staining. Some thoughts I have had is resting cells on a larger surface area (versus a 50mL conical), or straining again before staining (which will yield much fewer cells).
Thank you for your help!
Best,
Matthew
Typically, my group rests cell samples over night (O/N) at 37*C in 2mM EDTA/10% Serum/PenStrep/RPMI 1640 cell culture medium before analyzing phospho-proteins by CyTOF. Our Buffy Coat PBMC samples tend be highly sticky, as compared to normal PBMC. Even after a brief 25U/mL benzonase treatment, and strain through a 40um filter, the cells rested O/N will clump to the point that they can not be separated by pipetting.
I was curious if anyone has a good protocol for single-cell suspension of resting cells that might be a little more rigorous than what I have been doing, that works for phospho-staining. Some thoughts I have had is resting cells on a larger surface area (versus a 50mL conical), or straining again before staining (which will yield much fewer cells).
Thank you for your help!
Best,
Matthew