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293T cells not able to attach well after passage - (Jul/29/2018 )

Hi. I recently revived a 293T cells stock from liquid nitrogen, it did not look so healthy initially, but still I manage to grow it to full confluency and first passage I put 4 million starting cells number in T-75, it grows full confluency in 1.5 day. 

Second passage I put 2 million starting cells number in T-75 (viability was 96%), and when I observed it the next morning, I noticed the cells did not attach very well as usual. Many floating cells.

This experiment has repeated with other 293T stock and showed that 2 million starting seed is not good for cell attachment. and I will have to wait more time for it to grow into confluency.


But, I've been growing 293T for the past 6 months, and they always grow and attach well even I put 1 million starting cells during passage. 

Now, since they did not attach well and grow slower than usual, I do not dare to use them for transfection.


Anyone knows what might went wrong?




In general "If in doubt, throw them out" is the way to go, so I would do exactly as you suggest and not use them for transfection.


293 and derived lines (such as 293T) are well known for attaching poorly. There isn;t much you can do about this. They will still grow in suspension, but attachment is usually what you want to see. Attachment can be affected by quite a wide range of things in cell culture, but the usual suspect is the FBS. Make sure that your FBS is the highest quality and is tested batch-to-batch for issues such as you are experiencing.