[Cytometry] cause of cell clumping, apparently after Ab addition

Ruth Nissly rah38 at psu.edu
Fri Feb 14 14:32:04 EST 2014

A colleague is experiencing cells forming visible clumps in the antibody-staining process. I don't have any personal experience with the system she is using, so I'm in need of some advice. 

The researcher is using human blood (I'm not sure of the collection method). RBCs are lysed, then Dynabeads are used to remove CD45+ cells - the flow-through cells after magnetic selection are used. Cells are blocked with 2% BSA+5mM EDTA in PBS at 4C, then fixed in methanol (4C overnight). Cells are then washed, and primary antibodies are added in the blocking buffer; cells are at about 1-million/mL. The antibodies used are against cytokeratin 7, vimentin, and CD45. 

The cells are then kept at room temperature for 30 minutes for the staining, and it is at this point that cells begin to clump. It isn't clear to me whether the cells clump at RT even without the antibodies. 

I sense that it's something simple that I'm not thinking of. Can anyone enlighten me? 

Thank you! 

Ruth Nissly 

research technologist 
Microscopy & Cytometry Facility 
The Huck Institutes of the Life Sciences 
Pennsylvania State University 

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