My experiment involves pulling down cells on to a filter plate for a variety of assays. However, I want to ensure that my cells are alive so I am trying to resuspend them so that I can do an accurate cell count. However, I am unable to close off the mass-balance which suggests that the cells are either getting through the membrane or getting stuck.

Any suggestions on the appropriate way to fully resuspend cells? I'm currently using a P200 to mix the solution.

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    $\begingroup$ Have you tried trypsinizing them? Depending on whether they're suspension or adherent, and the time they've been on the membrane, you might be able to detach them just like you would from a culture dish/flask. Also, are you using a vacuum to adhere them to the membrane, or just allowing them to settle? Too much pressure may be getting them caught in the pores, although theoretically the pores should be much too small for them to fit through... $\endgroup$ – MattDMo Feb 13 '13 at 14:47
  • $\begingroup$ @Mattdmo, these are suspension cells so trypsinization will kill them. I'm using a vacuum manifold. I'm fearing the same thing which is why I'm doing this experiment to see if I can complete th mass balance $\endgroup$ – bobthejoe Feb 14 '13 at 23:09
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    $\begingroup$ Resuspending is hard due to attachment/entrapment in the filter and liquid retention around the edges. Consider using optical live/dead staining on the membrane? If you are concerned about pass-though you can also try and capture the fluid that comes through the membrane and give it a hard spin in a centrifuge. Look for a tiny cell pellet on the outside bottom (mark the caps). Or a simple and cheap dye for viability or counting that would be great on a filter $\endgroup$ – PlaysDice Apr 9 '14 at 21:42

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