Dear Researchers,
I am transfecting the insect cells (SF9) with the bacmid DNA to generate the baculovirus in six well plates. Normally Invitrogen protocol says that after 24 hours, cells would become bigger in size and more granular and after 72 hours, the cells will detach from the surface of six well plate. And one should collect the supernatant at that stage. But my transfected cells do look bigger in size and more granular as compared to uninfected cells but they are not still detached from the surface even after 5 days (some of them have detached but not all of them).
If anyone is working on baculoviruses and experiencing the same problem, could you please share your experiences? What do the lysed cells look like after they are transfected? Would all cells be detached after 72 hours or should I collect the supernatant medium from six well plates after all cells would be detached or will it work with only few cells detached?
I would highly appreciate your suggestions.
Thanks
Ruptured and bulged cells are mostly seen with aberrant refractive index and broken pm. You will also find few cells dark nucleated and semi round to elongated cells with different expression. If you keep all cells after transformation in selective media, round cells will die with in 8 hrs of time and you will get only your selected cells or cell clones
The transfection greatly varies according to the concentration and method of bacmid DNA isolation , if the bacmid DNA conc. is low , the signs of transfection is hardly seen and would take more time to detect , but as long as you mention that cells seems bigger and granuler after 72 hours , i recommend you :
1- To collect the transformed cells after 96 hours , freeze and thaw them for release of intracellular viruses , centrifuge and use the supernatant for infection of new SF-9 cells
2- If this don't work , try to prepare high conc.bacmid DNA using Midi or Maxiprep , and make new transfection trial.
You can also check my publication for more photos and description of the methodology
Good Luck
Mohamed Gadalla
Article Insect Cell Surface Expression of Hemagglutinin (HA) of Egyp...
Many thanks Mohamed. I actually collected supernatant after 7 days. I examined the cells under microscope and cells had a very rough morphology and they looked lysed. Is it right that I collected supernatant after cell lysis? Or 7 days is too long time to collect the virus? Any insights would help. Thanks
Dear Archit , the Baculovirus CPE is very characteristic , cells appear significantly bigger than non-transfected cell control , Granular (Large polyhedra) , Opaque or darker in color and later there is signs of cell lysis. I think there is no problem for longer incubation especially after transfection which already need time.
Anyway , try to infect new cells with the collected culture supernatant , if your transfection is successful , the CPE will appear within 4-5 days post-infection , once you see it again so proceed in preparation of P1 & P2 stocks and virus titration.
If available you can send me a photo for what you have under microscope to make the picture more clear
Many thanks again Mohamed. It was very helpful. Unfortunately, we don't have a camera fixed with the microscope. I will follow your suggestions.
Hi Archit,
Here are few things that I would double check during insect cell expression of recombinant proteins.
1) PCR conformation of the bacmid containing insert.
2) Testing at least 2 positive bacmids for expression
3) Isolating fresh bacmid just before transfection (or at least avoid multiple freeze thaw).
4) Infecting healthy growing cells, too old passages may be hard to infect.
5) And the most important is how you incubate the cells after infecting them with the bacmid. We found that infection is optimum in 24 deep well cell culture plate when cells are shaken at 200-250 rpm. Static protocol never gave us nice infection. This is probably related to your question. I suspect in static plates, cells on the upper layer gets infected while lower layers remain uninfected and therefore hard to detach.
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