Sf9 cells

I am starting to culture Sf9 cells and am growing them in Grace's Insect
Medium, supplemented, +10%FCS + gentamycin. According to invitrogen it
should be possible to slough the cells off the plastic when it's time to
passage them. But the cells I have stick like crazy and do not want to
come off that way. Tapping the flask does not help. I've tried
scraping them off gently but the viablity (by trypan blue) is not great
65-70%). I would like better viability before going on to the virus
infection step.

Any suggestions?

TIA
Allison

Allison <[Only registered users see links. ]> wrote:


Strange. I worked wit h SF21 cells, and it was no problem there. And
they are derived from Sf9, as far as I know.


Try to digest the cells for 5 minutes with trypsin. Remove the trypsin
afterwards and try again to tap them of. Should work now.
For how long did you culture your cells before you got this viability?
Maybe another medium works better, we tried Grace's and TC100 and
TC100 worked better.

Christian

--
Immerhin ist die relative Fluechtigkeit des Mediums Computer dem Gehalt vieler Bilder
in idealer Weise angemessen, waehrend man bei Papierbildern immer ueberlegt, ob "es das
Bild wert" sei. Oliver Corff, 23.08.05 in drf

Christian Praetorius wrote:

Trypsin was next on my list of things to try. thanks for confirming.

Someone else in the lab got cells from Invitrogen. She grew them to
passage 10 and froze them, and this is what I started with. I'm only up
to passage 12. But she also had problems with them sticking, pretty
much from the start I think.


Thanks for the suggestion.

Allison <[Only registered users see links. ]> wrote:


This is no problem. We used the cells for weeks without any problems.
Viability was usually up to 98%. Do you need to use adherent cells? We
also cultured the cells without any problems in suspension culture
(erlenmeyer bottles). For this, we added lipid to the cells and shaked
them around 70rpm.

Christian

--
Immerhin ist die relative Fluechtigkeit des Mediums Computer dem Gehalt vieler Bilder
in idealer Weise angemessen, waehrend man bei Papierbildern immer ueberlegt, ob "es das
Bild wert" sei. Oliver Corff, 23.08.05 in drf

In article <[Only registered users see links. ]>, Christian Praetorius <[Only registered users see links. ]> wrote:

It's oposite. Sf9 is clone of original Sf21.


Sf21 are more prone to shearing than Sf9. Early passage
cultures also attach quite strongly. In my experience, anything above
~ 50% viability when taking them off from dishes/flasks is entirely
acceptable and quite normal.


Grace is richer medium and in suspension cultures cells generally rich
higher density in grace than TC100.

DK