For sub-culture, rinse the remaining confluent adherent cells twice with PBS, aspirate the PBS, add 4 ml trypsin-EDTA into T75 flask and incubate for 4 minutes in the CO2 incubator at 37 °C.

After cells are detached from the culture flask, add 3/4 ml of culture medium, spin cells at 180 x g for 5 min, discard supernatant and add 1ml fresh media and repeated pipetting to make single cell suspension. use more 3 ml fresh media to adjust the volume 4 ml and seed in T25 flask .

to inform you if the T75 flask cell is 100% confluent with cells then you can split the cell in 3 T75 flask or more.

Masuma has given you the perfect answer above. If the T75 is fully confluent you can generate 3 T25 flask out of this one (T75)

Good Luck

Dua,

Please keep in mind that a T25 flask has 1/3 the growth surface area of a T75 flask. If you split the contents of your T75 flask into 3 T25 flasks there will be no room for growth.

Final volume in conical tube (after trypsinization) divided by 3 = volume per T25 for 100% confluence. Divide this volume by the desired split ratio (1/3 or 1/4 or 1/10 etc) to seed the T25s.

Example: The contents of a T75 at 100% confluency in conical tube = 6 ml

If you transfer 2 ml into a new T25 it will be at 100% confluency (no room for further growth)

If you transfer 1/4 of the 2 ml (0.5 ml) into a new T25 the split will be 1/4 and there will be room for growth.

With this example you would have sufficient volume to seed 12 T25 flasks. If you want only 2 flasks you discard the remaining cell volume.

If you don't want to waste media you can decrease the final volume in the conical tube. If you start with 3 ml final volume you simple take a fraction of 1 ml to seed each T25 (0.33 for 1/3 split, 0.25 for 1/4 split etc.

I hope this helps clarify your options!

Ellie

I agree with Ellie 100%!

I would ressupend the cell pellet with 4 mL media, take 0,5 ml into a T25 flask anad freeze all the remaining cells. You can have 3-4 criotubes.

Hello, Dua'a Yamani

1. for the first question i wanted to mean 3 ml or 4 ml.

2. add 1ml fresh media then 3 ml media in the conical tube then seed the 4ml in T25!? it is because after centrifuge your cell be in pellet. you do need to make single cell suspension where small amount of media can mix suspense uniformly then adding more media make suspense uniform cell

3. Actually you can do in two T25 flask . then the seeding cell density will be more than seeding in 3 T25 flask or more.

Thank you

Dua,

What you suggest will work but the new T25 flask will contain a lot of cells and be nearly confluent. You will likely have to split them again the next day. A confluent T75 flask will contain 3X the number of cells as a confluent T25. If you want to end up with a 1/5 split in the T25 you can resuspend the T75 cells in a final volume of 3 ml in your conical tube. Then transfer 0.2 ml to the new T25 flask (1 ml divided by 5). Add sufficient media to the T25, 9 or 10 ml. The remaining 2.8 mlst of cells in the ube should have sufficient cells to freeze 4-5 cryovials of cells. If your freeze media is 2x you can add ~2.8 ml freeze media to the remaining ~2.8 ml cells, mix and fill cryovials, 1 ml per vial.

Ellie

If you just want to propagate the cells from a 75ml flask and do not require single-cell suspensions to work with, then a simpler way is to just scrape off the monolayer in the 75 ml flask using a sterile 'rubber policeman' and repeated sucking-up-and down with syringe or pipette (titration) to segregate, before reseeding into smaller fasks. For sensitive cells, eg. stem cells, and primary cultures, proteolysis by trypsinization is not desirable and introduces a foreign protein as well. For serially propagated cells then trypsinization to propagate is ok.

If you want to be consistent, the best way is to count your cells after you have trypsinized them and stopped the trypsinization reaction and split them by cell density. Typically, we would split most cells to 1e4 cells/cm2, so 2.5e5 cells/t25 cm2 flask, but you should adjust the cell density based on your cell type to reach confluence when you need it to.

I know it's easier to split by ratios, so if you want to be consistent with your 75cm2 flasks, add 1/3 of the inoculum that you would add if you were splitting to your t75. Once you're in the t25, use the same split ratio as you had done previously.

I think, you could collect and freez cells from T75 flask then add media + FBS to the flask ( always a limited number of cells remain in the flask that thay can grow).