- I expanded my A549 stable cells from 12 well plate to 6 well plate should I freeze them down or expand them more in 25cm flask! ?
Because I read:
Some clones may lose expression after several passages. Freeze down samples from early passage to prolong their use after thawing
-IF im going to freeze them down should I use complete media or not with 10 percentage DMSO1! ?
Dear Doaa
I think you need to expand them shortly in 25 cm flask. After that, you can freeze down . Regard
In my doctorate studies, i expanded cells for 3-4 passages then froze in complete media with 10% DMSO.
Thank you all I read also in one of the protocols:)
All clones that you are interested in keeping should be passaged from the 6-well plate into at least 3 dishes: one for freezing as Passage 0 (in case something goes horribly wrong in the future you can always go back to this one), one for further passaging (if the line is already clonal),and one for sub cloning (if required).
Thanks for Your helpful a dvices last question
Should I freeze the stock cells in - 80 °C or in liquid nitrogen if im going to leave them just for 3-4 weeks ...
Usually If im going to freeze down my normal cells for short periods I STORE them in -80 °C ...
Another thing you could do is to slow them gently. We used to use a plastic container from Thermo called Mr Frosty™ that had isopropyl alcohol on the outer walls. We first stuck the cells in the -80 for a day and then into liquid nitrogen. Alternative is to construct an box in-house, we've had successes with improvised boxes.
https://www.thermofisher.com/order/catalog/product/5100-0050
Start to freeze your cells from around 3rd passage and possibly try to freeze them at every lower passage number to ensure you have a stock of healthy cells in case of any future contamination. Usually 8:2 ratio of FBS:DMSO is used for freezing the cells. For short term you can store them at -80°C but for long term store in LN2.
Another bit of confusion
If I want to do counting for the number of cells after centrifugation and at the same time I should responded the pellet in the selection medium for distributing the in different vessels.So if I want to do so can I re suspended the pellet for example in 1 ml complete medium then complete the media after counting based on the resulting number of cells AND no of vessels in the remaining amount of complete media diluted with the antibiotics!
Hi Doaa,
You yourself answered your question. You are right, just remember to use the volume of media for re-suspension based on the pellet that you get after centrifugation. Lower the pellet, lower the volume of media and vice versa.
I want to be sure because this time pellets are re suspended in selection not complete medium..I used to prepare selection medium volume randomly but now I wanted to do it based on number of cells after centrifugation..
Dear all I have another question regarding thawing stable A549 cells...
After cells being frozen in -80c for nearly 3 weeks I tried to do thawing and centrifugation for stock tubes but I found them most if not all died...
And I have done thawing for one of the stock tubes without centrifugation few cells survived..
I put antibiotic directly(immediately) in the medium without waiting for couple of days does it has any effect on cells!
So from your previous experience which method is better for recovering as much as we can from of those healthy cells from the freezing process!!
Dear Ruth thanks for your continuous reply:)
I have done what you recommended...
1- Usually Im warming up my vials at (RT) In order to avoid contact with water in the water bath...
2- Regarding stable clones I transferred them straight to selection medium and re suspended them in either selection medium immediately without waiting for a day or couple of days in complete medium then change to selection medium as few recommended that I dunno if this for particular cell lines or general rule till cells become activated gain after freezing process!!!.
Doaa Yamani
Dear Doaa
how did you thaw your stable cells finally?
did you add selection antibiotic directly after thawing? which method had the best result for you?
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