I'm trying to culture SKOV3 cells with RPMI 1640 medium+10% FBS+insulin+PenStrep. Something unusual happened after my first splitting with 1:2 ratio using Trypsin & EDTA. The shape of the cells looked different from the moment before splitting. After I split them, they had some kind of "line" shape and didn't have so many "pseudo-arms"/juts of cytoplasm for attachment compared to pre-split cells. I changed the medium on 25th Jan and I noticed my medium more basic (strong pink color) after I did change the medium. On 26th Jan, I noticed several cells were lysis. It's getting worse on the day after. Can someone please help me to find an explanation of this phenomenon? Thank you in advance.
Firstly, during your splitting the cells, Trypsin-EDTA treated the cells for too long time so as to do serious demage to the cells, secondly, the cells may be contaminated because of your carelessness. Generally speaking, cell shape change means the demage to their cytoskeleton to some degree. Just for your reference. Good luck!
Hello,
Have you thawed them freshly or is the culture passed to you from another person who has it in culture for a long time?
It is known that after recultivation form liquid nitrogen some vials do not start growing very wel, just thaw a new one for another stock if possible.
On the other hand when they have been passaged >50 times also artefacts can happen to the cells, change shape, do not grow and death by unknown reasons. Thaw also a new vial.
Add only 1x Trypsion EDTA for around 5 min (up to 10 cell line dependent) till you see the cell layer is loose and a single cell suspension will built. Trypsinize the cells too long kill them, as Ying-Yu mentioned.
Last but not least, as you said the medium turned deep pink or purple, you should have a look on bacteria in the medium. I have made the experiance when it gets pink you may have bacterial rafts floating on the medium surface.
You will not see in the microscop, because you have focused on the cells several mm below the medium level. This happens form time to time, also PenStrep is no guarantee for clean cultures, many germs form our skin are not distrubed that much.
It could also be a contamination with mycoplasma, some turn the medium faster orange or yellow, others maybe turn it to the basic side. Control with a mycoplasma test kit.
I would recommend, ask people around you if they could give you a new flask or thaw a new aliquot form nitrogen. Working with such artificial cells is just wasting of time, you can not be shure if the effects are treatment dependent or just get a gain or loss of effect due to the artificial behaviour.
Best regards
I recommend you check for mycoplasma. A contamination can change cell phenotype and behaviour.
The best solution is start with fresh cells from another freezedown.
Good luck,
Nupur
Dear Khrisna Putera Buddhiman The recommended media for SKOV3 is McCoy's5A with 10% serum.
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