Flow cytometry analysis - aggregates? - Analysis of aggregates (Jan/25/2003 )
I have a question regarding gating out aggregates on a PI signal width vs area plot. Our lab is studying a novel DNA binding protein, and it appears that when we transfect HeLa cells with a mutant form of this protein, there is 2-3 fold increase in cells that we would normally consider aggregates. Furthermore, knocking out the protein via siRNA causes a 2-fold decrease in the number of aggregates. Is it possible that cells are able to get very big in the process of cell division, so that cells that might appear as aggregates are really just two cells that have not separated from each other yet?
I would greatly appreciate any ideas on this.
Have you tried actually looking at your cell suspension under the microscope? Flow cytometry is terrific for giving you analyzable numbers, but sometimes you need to take a literal look at the cells themselves. I'd recommend investing in a hemacytometer and a differential stain such as trypan blue or erythrosin B. Then you'll be able to get an idea of whether you have giant cells (I've seen them) or sizable clumps (seen those, too).