Good morning everyone,
I am currently working on 3T3-L1 differentiation. I seed the cells on a 6 and 12 wells plates, and after 48 hours of 100% confluency, I add my differentiation cocktail for 48 hours.
At day 2 after adding the cocktail, I get holes as you can see it in the attached photo!!!
Did you ever had this problem? do you know what is the cause?
Thank you for your help!!
Could you post a picture when you consider 100% confluence? I think that your cells were not 100% confluence and as you know, when 3T3-L1 cells begin to differentiate (turns round, so its size is smaller than before, reason why you can see holes)
Are your 3T3-L1 dying or you only see holes but your cells don't die?
What plates are you using?(wells number) what volumen of medium are you adding in each step?
If you say that after 100% confluence and you initiated differentiation, I think the cells were over confluent and some cells might be detached or washed off. In our case, we initiate differentiation after 75% confluence. Also, make sure to use tissue-treated plates to assure the full attachment of cells.
Rosa, thank you for your answer, I use this protocol since a while, and i always wait 100% confluent to be sure of the CIP so the cells can start differentiation. but this is maybe the third time the hole thing happens!!! I thought it couldnt be about the confluency ! couldnt find the problem!
Are you adding your media directly to your cells? Try pipetting against the side of the well (not letting the pipette tip touch of course). You may be pushing your cells off the plate.
Thank you Thomas for your answer, unfortunatly it couldnt be the reason, because I pipette gently and on the side of the well
That's a shame! Did you manage to solve the issue? Could you send a photo of your cells the day you seed them?
Hi everyone,I solved my problem with 3T3-L1 cells, it is growing and differentiating well now, it was a matter of cells, i ordered a new vial and since then everything is alright, thank you all for your help
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