CaPO4 transfection prevents trypsin activity - Trying to split after transfecting (Aug/18/2004 )
Dear Fellow Cell-Culture Buffs,
Has anyone else ever encountered strange results when trying to remove cells from a flask using trypsin after those cells have been transfected using Calcium Phosphate? I am using COS-7 cells for transient transfection and they normally come off my flask within 3-5 minutes of adding trypsin. The only difference in my procedure has been treating the cells with Calcium Phosphate and a protein encoded in the pcDNA1 expression vector. Day 1 - split 1:5 into small tissue culture flask, Day 2 - treat with plasmid and Calcium Phosphate, Day 3 - change media and transfer cells to 24-well plate. I know there must be a way around this besides mechanically scraping the cells so that they can be used to create stable cell lines. Please let me know if you have any advice! Thanks!
Are you providing EDTA along with your trypsin? I have similarly transfected CHO cells with plasmid DNA using the CaPO4 method, followed by a DMSO shock 4 to 16 hours later, and have had not trouble in detaching my cells using trypsin-EDTA plus 2 minutes storage in the 37 C incubator (plus prewashing with Versene).