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CaPO4 transfection prevents trypsin activity


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#1 wendetha

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Posted 18 August 2004 - 02:38 PM

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!
-Wendy[COLOR=blue][FONT=Arial]

#2 LKLee

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Posted 26 August 2004 - 11:01 AM

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).




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