- S2 Medium 10% heat-inactivated FBS + Pen/Strep then 0.2um-filter-sterilize?
- I thawed a frozen S2 cells at P3, removed DMSO, and grew them about 2-3 days and split them 1:10 (so they are now P4) in multiple flasks for future freezing.
The new flasks grew just a little bit, hardly if I may say. Over the weekend, I noticed a lot of cell debris, much more than usual and also some motile black spots (Brownian? They did not do directional motion at 600x). While the flask, in which I thawed my S2 cells, grew normally.
Different thing I did this time is filter sterilizing the medium. Could it be the reason? Or did I dilute this too far considering the cells were just thawed and only passed 1x?
Thanks!
We never split these cells 1:10, 1:5 at most., and pass them every monday and friday Also, after thawing we wash them twice with complete medium prior to plating in 5 mL of complete medium. DMSO is extremely harmful for these cells.
Since the cells grow ok in the initial flask where you had the thawed cells in I would assume that your P3->P4 split diluted the cells too much. With a 1:10 split we usually culture them longer than 2-3 days before splitting. At a frequency of splitting every 2-3 days I´d go for 1:2 dilutions as the cells have a doubling time of approximately 3 days.
We never split these cells 1:10, 1:5 at most., and pass them every monday and friday Also, after thawing we wash them twice with complete medium prior to plating in 5 mL of complete medium. DMSO is extremely harmful for these cells.
I am pretty sure, the cells were too diluted. Usually, I let them grow for 2-3 days before splitting 1:5. If they are 90% confluent a 1:10 split may be possible too.
Dear Christa, I never filtered the medium, which should be already sterile when you buy it. Also, for defrosting and freezing it again, I follow the protocol attached which is the recommended by the manufacturer. Maybe your 1:10 dilution is too much, but ideally you have to wait until the cells reach a specific confluency (specified on the manual - 6 to 20 x 106 cells/ml), so you would need to count the cells before spliting them.
S2 cells anyway do not live forever, I see after 4 months of work I always need to get a new aliquote from the freezer and defrost again. What I recommend instead of freezing P3, P4, etc, the first time you ever get the aliquote of S2 cells from Invitrogen, to freeze as many aliquotes as possible, so that you always defrost P1 cells or maximum P2.
PS. I could not attach the manual maybe is too big, so here is a link for you to download from my dropbox folder. https://dl.dropboxusercontent.com/u/20447933/S2%20Manual%20%28Simon%29.pdf
Thank you all for all the advice!!!
I will keep an eye on it! I will split it 1:5 every 2-3 days from now on. And next time I defrost I will wash twice also, instead of just once, then let them grow for a week.
Yes, I have P2 and P3 frozen stocks. I also normally never filtered but somehow I am concerned that my stock might have been contaminated because I never filtered them. But I put the supernatant the P4 flasks that I pooled in 37'C shaken for 2 days and they were relatively clear compared to medium. (The pellet I used for an experiment)
Has anyone of you experienced these motile black spots? I read they are actually Achromobacter? Are they visually distinguishable from Brownian movement of debris?
Again thanks so much!
Dear Christa I agree with all the comments about doing a lower dilution when you split (twice a week preferentially) and the recommendations on the DMSO removal. About the black spots I don't believe they are contaminants but cell debris instead. They can be visualized only when cells are too diluted.
Do you really need to use Pen/Strep? I pass them without the antibiotics and they grow very well, splitting 1:10.
I have never spotted those black spots in my S2 cells culture.
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