I am infecting 4T1 with a lentiviral library and need to sort the cells to recover the transduced ones. I am running into problems with these cells clumping even after filtering. I use trypsin with EDTA to get them off the dish, inactivate the trypsin with serum containing media, wash 2x with PBS+4% FBS and then filter. During the sort I end up with many cells being 2 stuck together. This is also a problem during passage. Anyone have any experience with these?
4T1 cells like to stuck together. From my experience, it is very important not to let them overgrow at more than 70-80% of confluency, also not to trypsinize them for too long (I am trypsinizing usually for 1-2 mins). When I used TrypLE instead of trypsin, it also helped.
Hey Dennis: Saw this and thought it might help. https://www.stemcell.com/dnase-i-treatment-for-clumpy-cell-suspensions.html
Also have you tried using Soybean Trypsin Inhibitor (in PBS) instead of serum to inactivate the trypsin? I use is routinely with our primary skin isolates. If you want to try it, I can give you some.
Also, maybe a higher concentration of trypsin might help (ie, 0.25% vs 0.05% + EDTA).
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