I am currently growing BICR31 cancer cells for conditioned media collection, and as I passaged it I have seen many bubbles/vacuoles - like structures. Cells were healthy during the first split (P8). First image is P8 and second is P10. Has anyone experienced this before, is this normal?
Hello Nur Mohd Faizal
The appearance of bubbles in culture is because of some kind of stress. Keep passaging the cells and after a few passage you will notice that the bubbles/vacuoles disappears. There are many factors that can cause stress. For instance, at the time of thawing or during trypsinization of cells.
Good Luck.
I agree with Malcolm Nobre - some cells do no like trypsinization and your cells appear stressed. Consider using TrypLE which is a good replacement (usually you do not have to change the protocol for detachment). I use Gibco: https://www.thermofisher.com/uk/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin/tryple-express.html?icid=cvc-dissociation-c2t2
I also think your confluency is too high in these images. As a benchmark for most cell lines, culture only to 80 % confluency (to maintain log growth phase), passage in a 1:3-1:4 ratio and replace media every 2-3 days.
Dear Nur Mohd!
These vesicles are most likely autophagosomes.
Article Lipid accumulation in human breast cancer cells injured by i...
Fig. 3 d
This suggests that the cells lack nutrients. Try changing media or changing media more frequently with serum.
thank you Malcolm Nobre , Jack Hopkins and Alexandr Chernov !
Malcolm Nobre When do you say during trypsinization, do you mean I left it too long in trypsin? I put 1mL trypsin and it took awhile sometimes. Or should i put more trypsin instead?
Jack Hopkins I'll consider that as well. At the moment I'm using 0.25% Trypsin/EDTA from Gibco too
Hello Nur Mohd Faizal
Trypsinization has to be done for a max. of 5 mins or less in the incubator. The volume of trypsin should be such that the cells are submerged in it. Tap the flask gently to dislodge the cells.
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