I had frozen my RAW 264.7 cells from T-75 flask having 80-85% confluency, washed with 3ml PBS respectively. After washing, I added 3ml of fresh media and scrapped all the cells (also, could we use trypsin for this cell, if yes then how?) and spin down @ 1200RPM for 5 minutes. Discarded the supernatant and again added the fresh media about 3ml and mixed uniformly. From the 3ml media containing cells, I added 3ml of Recovery™ Cell Culture Freezing Medium (Catalog number: 12648010) and made 6 vials and stored them at degrading temperature (Like stating from -20 to -196).
But for reviving the same cells, cells (RAW 264.7) aren't adhering on culture plates (per se T-25 flask).
So, suggest any right way to run my culture smoothly.
Medium used: DMEM
Thanks.
If they are not adhering, your cells are not viable.
You need to freeze your cells at a higher concentration. Usually 1 T-Flask per vial. Better chance of getting enough viable cells to reculture after being in liquid nitrogen.
@ Shen-An Hwang Yeah, I already prformed 1 T-flask per vial but the condition is still the same, it is not attaching to the surface. Also, what would be more convenient for detaching the cells from the surface either from scrapping or from trypsin treatment and how?
RAW is so hardly that you should be able to just scape them off the flask.
Did you ever check the viability of the cells after you scrape them?
You can certainly try Trypsin/EDTA. I've always just used the ones from Sigma (0.25% Trypsin EDTA). Usually just 10 minutes at 37C is good.
I can't speak about your freeze down protocol, because I am very old school. My freezing medium is 90% or 95% complete FBS with 10 to 5% DMSO into each cryovial. Then that goes into a dry ice isopropanol bath. After about 15 minutes, I transfer that into liquid nitrogen for storage. Never had a problem.
One last thing... when you regrow the cell from the frozen stock, you spin down the vial right? So none of the freeze down medium goes into the cell culture?
The cells shoould stick over night (or >4 hours). Usually, at this time, I pour off the medium and add free new medium. This gets rid of the dead cells, and allows the remainnig viable cells to grow better. Don't leave dead cells in the flask, they tend to just cause other cells to die.
That's all I got. Hopefully something in there will help you.
I have already tried with spinning step as well directly transfer of cell to a fresh new media in T-flask while reviving the cells from cryo-vials.
The cells didn't attach in both the conditions.
If the cells are not adhere to the surface that means the cells are not happy. I am working with cell culture and I noticed if the cells are happy a few minutes after you add the cell into the flask the cells started to adhere and growth into the flask. I think you need to check if there have some cell adhere you just need to leave them to grow and if not you just need to use a new batch of the cell.
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