cells not adhering to flask after thawing - (Jun/13/2006 )
I always thaw my cells in my hands, then add a little warm medium, transfer into a Falcon and dilute. Centrifuge to get rid of the DMSO and resuspend into flasks. I never had problems with any of my cell lines. Give it a shot, it's even less work than changing medium after 3 or 24 hrs.
-britzelbeere-
QUOTE (mmini @ Jun 13 2006, 02:45 PM)
QUOTE (scolix @ Jun 13 2006, 02:04 PM)
After thawing the cells and seeding them in a flask, i change media after 3 hrs to get rid of DMSO, as its toxic.
thats a good pt....i didnt do that yet...i just tried to put them into another flask..but they might be dead anyways so that would've been pointless...i'll try adding trypan...thanks!
thanks so much!...i did directly put them into the flask and so i probably went wrong there....i got some new cells and they seem to be growing well now...thanks
-mmini-
QUOTE (fred_33 @ Jun 14 2006, 06:30 AM)
as rhombus said, it's important to dilute cells in great volume of medium (to dilute DMSO basically), spin an remove supernatant wich contains DMSO. After that step, you can put your cells in the flask.
You can't directly put cells from DMSO stock to the flask (that's i understood regarding your post, please forgive me if i'm wrong)
You can't directly put cells from DMSO stock to the flask (that's i understood regarding your post, please forgive me if i'm wrong)
thanks! that sounds great.....i will be thawing out some new cells soon so i will try that...thanks again
-mmini-
QUOTE (mmini @ Jun 13 2006, 06:41 AM)
yesterday i thawed out CHO cells from liquid nitrogen and grew them up in a t-25 flask (for thawing i placed the vial in 37C water bath until cells were thawed and then added them to a flask containing 5 mL of warmed media (DMEM & 10%FBS). Today i checked them and almost all of the cells are floating...none of them adhered to the flask...is there something I can do??
Hi Everyone
It's been a longtime accessing this forum and I see I have been missing out big time. Mimmi to also respond: try to increase FBS to 20%. Again which CHO cell are you using bcos there are different kinds (eg, CHO (protein free, CHO-K1/SF), make sure the one you are using is cultured in a compatable media, ie the ones I'm familair with need Ham's F12 media ref: www.ecacc.org.uk.
Ciao
Lyndza
-lnkabinde-
Have you held a memorial service for them yet?
We use 90% FBS/10% DMSO. Dump the thawed cells rapidly into a flask with 20ml of media.
-scientist-