Insect sf21 cell lysing in shaker culture...

Thomas Burmeister tbu at gmx.net
Wed Sep 2 14:02:51 EST 1998


In article <6s4q2u$e8e$1 at news.doit.wisc.edu>,
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote:

>Needed 250 ml suspension culture of sf21 cells. Normally
>I propagate them in flasks (~10-15 ml) or spinners (100 ml).
>
>Decided to try shaker culture. 250 ml in standard 1l flask
>at 27C in TC100 with FCS. Seeded at 0.3x10^6/ml. 
>Shaker speed was 140 rpm (as recommended). 
>Next morning there were NO cells at all. Opalescent solution,
>barely any particles are seen under the microscope...
>Btw, this was not virus infected culture. 
>
>Why and how??? My only guesses, both seem rather unlikely
>to me:
>
>1) Extreme sensitivity of these cells to shaking (yes, these cells
>are indeed more fragile than sf9 but they are perfectly happy in 
>spinners). 
>2) Dirty flask. Unfortunately, our lab does not separate  flasks for
>E.coli culture from all other glassware (I know, I know!). The flask
>was machine-washed, no acid, then I manually and very carefully
>rinced it with pure water before sterilizing.
>
>Anyone ever saw anything like this? I will certainly repeat it and 
>be more careful to eliminate #2 but I really cannot imagine anything
>that would be SO harmful for cells at such [expected] low 
>concentrations. 
>
>Thank you for any comments you might have.
>
>        - Dima

I have been working with Sf9 and some derivatives of them some time ago. I
did not observe extreme sensitivity to shaking or spinning. Are you using
antibiotics in your cell culture medium? I would strongly suggest to use
Ampicillin (100 mg/l) and Streptomycin, even if you are sure that your
medium and glassware is sterile (as should be).

Thomas Burmeister




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