we getting a rounding of the cells when we retire the medium and add new one. Sometime it happens but not always, especially when the cells are sown in coverslip. However, today, the cells were in a p100 cell culture dish, I changed the medium and add new one, i look at them in microscope and the cells gat a round shape. A curious thing is that after overnight, the cells recover and get a normal elongated shape.
we do not sow the cells, since in our laboratory, that is the function of the researcher assistance, but i realized that sometimes, the culture dishes are recycled.
Do you think this may be the problem? or Maybe the cells are contaminated or old?
The Coverslips are new, and still the cells get a round shape
Most cell types would be influenced by the mechanical disturbances caused by the drastic liquid flow when you change media. The nature of their adherence to the support would be necessary to know, are they epithelial/endothelial cells for example. Most of the standard eukaryotic cell lines would adhere better to culture plast, than to glass. Rounding is typically equal to loss of adherence, after the change they adhere again, as you observe. It may also be a pre-apoptotic event, this can be investigated using one of the commercially available apoptosis kits , for example http://www.abcam.com/kits/apoptosis-assay-kits
You probably need to tell us a bit more on what cells you're working with and what medium and conditions you use. Rounding of the cells can have various causes. Contamination can usually be seen through the binocular and your Population would show distorted growth. Could it be that maybe what's shown in the comic I added could be your problem? :-)
Most cell types would be influenced by the mechanical disturbances caused by the drastic liquid flow when you change media. The nature of their adherence to the support would be necessary to know, are they epithelial/endothelial cells for example. Most of the standard eukaryotic cell lines would adhere better to culture plast, than to glass. Rounding is typically equal to loss of adherence, after the change they adhere again, as you observe. It may also be a pre-apoptotic event, this can be investigated using one of the commercially available apoptosis kits , for example http://www.abcam.com/kits/apoptosis-assay-kits
Hi, si it possible that your cells have some problem with growing on coverslip? If the cells don't adhere very well you notice a rounded shape cells (the same aspect of trypsinized cells), do you have problems with the coating?
I agree with Birger and I think you are seeing a transient partial loss of adhesion since it sounds like it resolves. Knowing more about the cells will help. Is your media pre-warmed an is the ionic content/concentration appropriate? Any carryover of EDTA or chelator from recycled dished that have had Trypsin EDTA could perturb cell adhesion briefly, but unlikely the case here if you only see this at media changes. How do you wash (if at all) between media swaps? If you use Ca/Mg free PBS you may be washing out key ions necessary for adhesion molecules which are then in your subsequent media, hence the transient nature of the rounding.
As the others say, it sounds like a transient mechanical thing. Would your experimental conditions allow for, say, removing half the medium and adding half fresh, very slowly and carefully? That would also minimise changes owing to a different balance of nutrients before and after the change.
Have you tried retaining 25 to 50% of the previous media, add new media to this. This may allow the cells to retain original form and growth characteristics by reducing the shock and retaining growth factors in the original media.
Culture dish (plastic) or cover slip (glass)?
When I feed my cells (MDA-MB-231) they round up for about 4-5 hours and than they stretch again. We assume this a pro proliferation response induced by FBS (We are using 10% in DMEM). I cannot see rounding up if I change to OptiMEM (reduced serum) or serum free condition.
- Badges
- Science method