[Cytometry] cause of cell clumping, apparently after Ab addition
rah38 at psu.edu
Tue Feb 18 16:10:04 EST 2014
Thank you for the input from several flow-ers. My colleague will try using PFA to fix the cells - it seems likely that her current method is injuring the cells too much. She was hesitant to use PFA because she thought it would make the DNA of poor quality for down-stream sequencing, but she'll give it a try - SOME DNA is probably better than just a lump of dead cells.
----- Original Message -----
From: "Ruth Nissly" <rah38 at psu.edu>
To: cytometry at lists.purdue.edu
Sent: Friday, February 14, 2014 2:32:04 PM
Subject: [Cytometry] cause of cell clumping, apparently after Ab addition
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?
Microscopy & Cytometry Facility
The Huck Institutes of the Life Sciences
Pennsylvania State University
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