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Buffy Coat PBMC - Single Cell Suspension

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mkunicki

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Posts: 17

Joined: Wed Apr 15, 2015 8:43 pm

Post Fri May 15, 2015 5:31 pm

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
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reginaharley

Participant

Posts: 5

Joined: Thu May 15, 2014 1:40 pm

Location: University of Maryland - Baltimore, Baltimore, MD, USA

Post Fri May 15, 2015 8:08 pm

Re: Buffy Coat PBMC - Single Cell Suspension

Sounds like you might be getting some cell lysis which is causing all the free DNA to stick stuff together. Try a little DNase.

Regina


Regina H. Harley, M.S.
Lab Supervisor - Flow Cytometry Facility
Center for Vaccine Development
University of Maryland - Baltimore
685 West Baltimore Street
Bldg HSF1, Room 456
Baltimore, MD 21201
(410) 706-0095 FAX: (410) 706-6205
rharley@medicine.umaryland.edu
Core lab email:
cvdflowcore@medicine.umaryland.edu

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