I transfected Hela cells using PEI (1:3) with a GFP positive plasmid. Six hours post transfection the media was replaced with fresh media.
After 72 hours the transfected cells were harvested by trypsinization and then washed with 1x PBS.
The cell pellet (~10^6cells) was resuspended in 1 ml of FACs sorting buffer (1X PBS, 1mM EDTA, 25mM HEPES, 1% FBS, 1X penstrep, filter sterilized in 0.2um filter).
The cells were sterile sorted for GFP positive using 120 micron nozzle size. 15% of the cells were GFP positive.
The collection media was 20% FBS in 1xDMEM (the collection tube was pre-washed with FBS to avoid cells sticking to the wall).
I got around 5000 cells. I collected the cells washed them with PBS and resuspended in 200 ul DMEM media and plated the entire volume in one 96 well plate.
When I checked these cells under microscope all the cells were dead (I could see cells but they looked apoptotic). I kept the plate for 2 weak but still I couldn't find any growth what soever. So I concluded that all cells were dead.
Have anyone experienced similar problem? Is sorting too stressful for hela cells? or is PEI transfection accompanied by FACs sorting killing hela cells? What am I doing wrong?
Additional Information: Before this I have done single cell sorting of hela cells with similar process and collected the GFP positive hela cells in two 96 well plate containing 150ul of DMEM media. Even in this condition all my cells died.
FACS sorting does not kill HeLa cells, I had used this method to sort HeLa cells, NIH3T3 cells sucessfully. My protocol: 10^6 cells (expressing GFP fused recombinant protein) in a 10 cm dish was harvested by trysin digest, then washed three times with 1 X PBS and filtrated with a 300 mesh nylon membrane, then subjected to the FACS. The collection buffer was 1 ml 100% FBS in a 15 ml tube. 10^5 cells were collected and seeded in a 6 cm dish (the media was adjusted to 5 ml), after cell adherence, about 6 hrs, the culture media (mixture of 1X PBS, DMEM, high concentration of FBS) was replaced with newly warmed, high glucose DMEM, with 10% FBS and pen/strep. The cells then grew well.
I think our sorting methods are similar, the potential problems may be: 1. the cells you collected were too few, only 0.5%; 2. after sorting, additional processes should be avoid, such as centrifugation, since the cells had suffered for a long journey (washing with PBS needs several rounds of centrifugations); 3. the cells should be seeded in a 24- or 6-well-plate since the 96-well-plate cannot support the growth of so many cells for such a long time.
Good luck!
Thanks for the response Chen. Will incorporate your suggestions in my next experiment. did you use transfected Hela cells? if yes then what was your transfection reagent? do you think transfection reagent is important in this case?
We use Lipofectamine 2000 for HeLa transfection. I don't know whether transfection reagent has an influence, but PEI was indeed much more poinsonous to cells than Lipo, but it's cheap.
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