I joined a new lab and I have to start work on 4T-1, but its not reviving. I don't understand, we are using RPMI 15%. My new lab mates freezed the cells and after adding freezing media they kept the cells for 24-48 hours in -20C, then they shifted cells to liquid nitrogen...
Hello Priyanka,
If your cells are not reviving, means there is some problem with the freezing protocol or procedure in-between. Make sure you are doing everything right. As per the storage protocol is concerned, Keeping cells in -20 for 24 to 48 hours is not recommended. Its always good to keep them in -20 for 2-3 hours then @ -80 for the same time before shifting them to liquid nitrogen. It is always good to decrease the temperature gradually. We keep cells with freezing media @ -80 in a isopropanol box or in cotton for at-least 24-48 hours before shifting to LN. 4T1 revives very well if stored properly. We even use our 3-6 month old -80 stocks, they also revive well. Remember, once DMSO in contact with cells, work quickly and ideally get the cells at -80 within 3 min of contact.
In my opinion you should try some old stocks or if they dont work, its better to get new cells from some other source.
All the very best
Thnax Ravi for your answer.. We don't have old stocks, whatever storcks we have are made from same protocol.. Is any body is there in your knowledge working on 4-T1 and can give me.
Do you use antibiotics? We are culturing 4T1 in DMEM with 10% FS, no antibiotic
Yes, I am adding Pen-Strep,.. Ok..I will use DMEM 10% FBS with no antibiotics...
I think the best to use the media, to which cells were adopted before without antibiotic
I find re suspending the cells in a 1:1 media and FCS solution is best. Then adding drop wise a media DMSO solution (1.5ml DMSO to 8.5 media). Put in -80 for 24 hours, then move to liquid nitrogen
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