Hi

I just started working with baculovirus expression system for protein expression. Iam growing Sf9 cells in tnm-fh media. Recently i have been observing strange black particles outside the cells ( in the medium) which seem to increase with time, gets carried over with centrifugation. It dosent seem to be bacterial contamination as this is observed even with antibiotics (just gentamicin or gentamicin-cipro combination). It is also observed in just the medium without cells after few days. It dosent change the pH of the medium nor does it make the medium turbid.

Is this media precipitation? How can I overcome this problem? Do anyone have any idea?

Thanks in advance .

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Dear Kalps,

Some cells release this debris into the media. Sf9/Sf21 do this and it will not affect the cells ability to be infected with baculovirus. Other cells that do this are DLD-1 and Caco-2.2

My tips for growing these cells:

Use a cooled incubator.....i.e. LMS
Grow the cells in Techne Stirrer bottles....specifically designed to grow suspension cells.
Keep the cell density between 400,000- 800,000 cells/ml
USE NEW ZEALAND ORIGIN FCS/FBS...the best quality serum will increase your protein expression by between 50-90%
Grow the cells at 23 degrees centrigrade
Infect at 28 degrees centigrade and infect in adherent cells.
Every 2 months get new cells out from frozen stocks
REGULARLY SCREEN FOR MYCOPLASMA'S.....agar growth assay/Hoescht staining.

Hope you find this useful.

Kindest regards.

Rhombus

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Thanks for the useful tips. is there a way to remove those debris? like using lower centrifugal force or something like that? Because i have to remove it from the culture i started from my last master stock. I use 1000 rpm in an eppendorf centrifuge to pellet cells.

Second, thinking it to be a contamination, i started growing the cells in 40micg/ml of gentamicin. Can i stop using that?

Does low temp like 23 help in preventing formation of this debris?

regards
kalps

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Dear Kalps,

Lowering the temperature will not affect the debris formation.

I do not like using any antibiotics/antimycotics at all in any of my cultures....they mask:

Bad technique
Bacterial/fungal cryptic contaminations


....and are dirty compounds i.e. will have many other cellular effects

Kindest regards

Rhombus

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