Hello, I thawed/plated one frozen iPSC vial and though I saw cell attachment during the first 24-28 hours I noticed after that point the colonies began disappearing. This is in matrigel coated dishes with E8 media. I also noticed these strange "bubble" looking objects in the culture (photos attached). I am wondering if this is mycoplasma or unrelated to the cause of the iPSC cells detaching. Both are 4x mag. Thank you in advance.
Bubble objects looks like water/ethanol on the bottom of petri dish. Definitely it is not mycoplasma. Before imaging remove ethanol/condensate from bottom of petri dish with dry wipes and check whether your microscope focus is on the cell culture surface and not in the bottom of plastic.
And your cells dying not because of mycoplasma.
Do you use ROCK inhibitor?
Hi Vikrant Sharma,
I agree with Alex Mitriashkin, you are not focused on the cells. Even when there are no cells you should be able to see the matrigel and use that to focus.
Unfortunately, with these images we can't really help you as one cannot even see dead cells floating.
Best wishes
Selene
Hi,
The image is slightly out of focus, so it’s difficult to be certain — these could be small water vapor bubbles on the lid, single very round colonies, or contracted floating aggregates. If colonies “disappear,” they may in fact be small, low-viability colonies detaching from the substrate, contracting, and forming 3D aggregates. To improve survival after thawing, use tissue-culture–treated plates coated with fresh ECM (e.g., Matrigel, rh-Vitronectin, Laminin-511), thaw colonies in the presence of ROCK inhibitor (10 µM), and handle them gently to avoid breaking colonies into very small fragments.
Good luck!
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