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Hela cells clumping in center of well of 96well plate. - (Mar/17/2006 )

I am using HeLa cells for a 48 hr transfection and I use a Multidrop to dispense the cells into a 96 well plate.
First in the well is 30uL of transfection mix that is serum free, to this I add 30uL of HeLa cells at a concentration of 100,000 cells/mL with 20% serum so that final serum conc. is 10% (final well count is 3000cells/well in 60uL total).

this is incubated for 48hrs, and what I'm seeing after a day or so is that the cells are unevenly distributed in the center of the well. Has anyone seen this before and how did you fix it? My end point is fluorescence imaging so I can't have a monolayer and it helps if the cells are even distributed instead of clumpy.

I use Trypsin to harvest the cells for about 4 minutes or so, and I do tap the flask to loosen the cells, (which I know can contribute to clumping) but I pipet up and down to get an even suspension. When I plate using the multidrop I am using a stir bar. I'm running out of ideas to try and troubleshoot this! help?

I spoke to a tech support rep at Perkin Elmer today who suggested that my low number of cells in the well initially is contributing to them gathering in the center of the well. He suggested that I lower the volume of media in the well, which would increase the concentration while keeping the number the same. does this make sense? would it help to make the cells more distributed?

thanks so much for taking the time to read


a friend of mine had a similar problem, and we pinpointed it to the technique - are you touching the bottom of the plate when you eject the cells from the mutlichannel ?
Pippetting into the media and swirling a bit really helped to reduce clumping
hope this helps