Sf9 cells culture conditions

[tavernier7]

Hi,
I have to cultivate Sf9 cells to produce recombiant proteins after baculoviruses infection.
My question concerns culture conditions of this type of cell. Please, can you indicate me how do you cultivate them… I have no experience regarding these cells. Actually, I try to cultivate them into the Lonza Insect-XPRESS™ Protein-free Insect Cell Medium with L-glutamine, using TPP plastic 75 cm² flasks with closable lids. Cells are low passage (P6) and come from a lab in Pasteur Institut. In fact, I’m cultivating these cells exactly as in the lab in Pasteur. And cells don’t grow : when I split them (1/5), new batch do not grow…
I’m sure that I’m in sterile conditions as no trouble can be observed in flasks.
What can you advise to me ?
Thank you very much in advance, best regards.
Nico

[protolder]

Hola Nico, I didn´t realize of this mail and I have discovered today. Well, this medium is for serum free medium adapted cells as the Sf900 from invitrogene, they are designed for grow at high densities in liquid medium but they grow in bottles too. The most conventional medium is modified Grace´s (TNMFH) with serum .In it, cells grow in monolayer and suspension in spiners, because if you want grow them in baffled flasks you have to add Pluronic 0.1% to protect cells of agitation forces. So you have to look in what medium your cells have been grown, and seed them in it or similar. After when you have good grow, because they grow very well,if you want you have to sequencially adapt them to the new medium About your last question of DTT in Kinase assays, here we do them cdks, PIM family PI3ks and i asked to my collegues with do them, and say me that without reducer, the activity is lower.