Insect sf21 cell lysing in shaker culture...

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Thu Aug 27 18:29:10 EST 1998


I would appreciate any comments on the following:

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



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