I have been work with cancer cell line. As I subculture it more than 3 times moving black particles appear in it, and while adding trypsin it takes a longer time to detach. Sometimes it does not detach and cell line undergoes apoptosis.
Please help me.
Thanks in advance
some times just type of particles (if I understood you well) comes from theater. Sometime it is contamination. Try other than trypsin and try to see.
I hope I was of help to you.
1. Is your media getting turbid, then its a contamination.
2. If its not a contamination, then prepare new medium and take out a new vial of cells for culturing.
3. Try to use a lower percentage Trypsin like 0.05% or use tryple E to avoid apoptosis.
Hello,
Thanks for every suggestion
My media is not turbid and I used antibiotic as well and new media. But I still don't understand , when I start with cell there is no particle but as I subculture it 2-3 times it stick strongly with surface and do not detach with trypsin. and of course there is lot of moving black particle.
I would argue against the use of antibiotics in culture of established cell lines. I stopped using P/S 10 years ago and had no increase in incidence of infection.
These black dots that keep moving are primitive archea bacteria. I have successfully eradicated them with BM cyclin from Roche. Good luck!
You can get rid of these particles, by growing the cells as xenografts in NOD/SCID mice and doing primary culture of the tissue grown. But it is tedious.
Ruby
Dear Richa,
My friend, those cells are contaminated. I have seen this several times. One challenge in the cell culturing technique that every scientists face is the issue of contamination. As a matter of fact, the sensitivity and the high propensity of most cells lines to be contaminated is exactly what makes this technique a challenging one.
Another issue that complicates this technique is the resistance of some of these bacteria to antibiotics. When you encounter heavy infection, it must have been due to the high resistance of the bacteria to your antibiotics. Do you use Penicillin and Streptomycin antibiotics? The synergistic effect (Beta-lactam inhibition and protein synthesis inhibition) would help kill bacteria, 1% should be Ok in the medium.
Another issue which cannot be undermined is your technique. You might have contaminated the cells yourself while culturing them. You need to prepare for calculated steps while culturing cells.
Some may advice the addition of antibiotics into the flask. Well, personally, I dont see that as a professional idea but it might work for light infections but when the bacteria infection is heavy, I would only suggest you discard them because they would infect other cells in the incubator. I am so sure you wouldn't want to experience that.
Best Regards
Adelusi TI.
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