Hi,
I am having some trouble to do Focus formation assay in NIH3t3 cells. I start with making NIH3t3 cells stable using retrovirus (90% infection efficiency after 72hrs with pSIR-GFP virus).
My trouble is when I allow the cells to grow for 2 weeks, they develop some clumping contamination just after 3-4 days (after becoming confluent). Even though I change media after every 2 days (DMEM+10% FBS) to replenish the cells, but this contamination (large clumps , brownish color , floating) comes in 3t3 cells only. Transformed or non-transformed both are affecting 3t3 cells. Can someone please suggest any other type of media or growth conditions for doing this assay.
The morphology of cells seems correct.
Thanks
Based on what you describe, you might have got fungi in your cultures. If you don't have antifungals in your medium (like amphotericin B), and if your cells are not irreplaceable, I would recommend to discard your current cells and to re-derive them in a such medium. If you already have antifungals, then re-start with a cocktail of different antifungals
The other possibility is that the density of your cells is to high. 3T3 cells tend to de-differentiate in culture and to grow in suspension ( clumps) if they are allowed to overgrow, or if they are too much diluted at passages. Ideally, cells should be passaged twice a week at 2 to 3 fold- dilution maximum
Dear Barban
Thanks for your response. I checked correctly it dos not look like fungal contamination. However in few articles I read that for Foci formation assay u need to allow the cells to grow for 2 weeks in 4% FBS containing DMEM instead of regular 10%. I am testing a small dish of cells in low serum media, hope that grows fine.
If you come across any info regarding antibiotic selection of NIH3t3 cells with Puromycin & Neomycin, pls share with me :)
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