I have infected my insect cells (SF-21), with P4 viral titre. Now the protocol suggest that it should be harvested before lysis. Can anyone suggest me how I will figure out when to harvest after cells or my cells are ready to harvest.
Thanks in Advance.
Prem
You don't need P4., baculovirus acquires deleterious mutations with more passage. Usually, P3 is the best titer.
SF21 double every 24 hrs. Optimal harvest times can vary for each baculoviral construct. You can check under the microscope to determine the best time for collection. You can make small culture to test the time like 24 hrs to 72 hrs. You can also do western blot
Mahesh Chandak What morphology I could expect under the microscope ? So you mean if the cells are doubled that means it has expressed the protein inside the cell ? I am totally new in this field so just confirming what could be the practical difficulties one can face during the optimization of harvesting period.
Thank you for your suggestions
Prem
Use inverted phase microscope like 250–400X. Usually, around 48 hrs of infection there is granular appearance to the cell and they stop growing. You can see change in shape of cells like 25 to 50% change in diameter or nuclei looks like they fill up cell. I used SF9 cells after infecting them I usually see that they
become elongated or nuclei fills up cell - For my virus it took 48 hrs for sf9 cells and I got good yield of protein. If you overgrow cells extensively then they will lyse in pieces.
Prem Prakash I have used Tni cells for protein over-expression and observed increased cell diameter with cell debris around after 48-50hrs of P4 virus addition.
Mahesh Chandak Could you share Sf9 cells infected with P3/P4?
Thanks a lot!
I don't know what you mean by " share sf9 cells infected by P3/P4". I hope you mean a picture. If so, then sorry, I don't have any soft copy to share.
Mahesh Chandak Yes, I meant picture of Sf9 infected with P3/P4 virus titer
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