these photos taken by phone for cells at day 4 and day 7 culture. shall i transfer that in picture 2 to bigger t-75 flask or not yet?
I'd say 30-40% confluent. Wait till it reaches 80%. Then probably transferring to a T75 will be more practical. (T75 when fully confluent can yield around 7-8 million cells.)
Definitely less than 50%. I would put fresh media on but not split yet.
Generally every 2-3 days put on new media even if not splitting.
thanks. i put media when change color to yellow or orange. sometimes, daily.
I'd say 30-40% confluent. Wait till it reaches 80%. Then probably transferring to a T75 will be more practical. (T75 when fully confluent can yield around 7-8 million cells.)
Hi Heba,
Looks to me approximately 40%, change media every 3 days (earlier if media changes to yellow). I would say keep feeding until it reaches 80%, definitely don't sub-culture yet as some of those cells do not look like they have spread across the surface of your culture plate yet and splitting your plate will put too much stress on the cells.
I have attached a good PDF from Abcam outlining when to sub-culture cells. Hope it helps.
Sam
hope this 80% now. i had to transfer to bigger flask anyways as tomorow is the weekend.
That is about 50%. What kind of cells are those and what kind of media?
I would not split but I would change the media. These cells are growing slowly. They almost look like fibroblasts but hard to tell with that picture
do you mean first picture peter or picture in comment? i think it is because i originally seeded less number of cells. for fully confluent T-75 flask, how much medium do u dilute the pellet after centrifuge and how many 1-8 crovials do u use for freezing one flask?
For a T75 the usual operational volume ranges between 20-30 ml. Each cryovial should have 1-1.5 million cells. A full confluent T75 should have 7-8 million cells. So I'd say 5-6 1 ml cryovials can be frozen down.
I agree completely with numbers presented by Sumit with regard to freezing. Follow those recommendations. I would use a commercial freeze media if the cells are special ( hard to get) like Frostlife.
With regard to splitting. Primary cells are different than transformed. They do not like to be overly sparse or overly dense. In general, this is empirically determined by the investigator. In general, 8,0000 cells / cm^2 is going to be in the order of 8-10% confluent for most cells.
Thanks . So how much medium to add to the cell pellet? I add around 8 ml with 800 ul DMSO
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