Hey guys,
I need your help to decide if I should include floating dead cells in my western blot analysis. I am looking to check expression of certain proteins after drug treatment in adherent cell culture.
My question is after the drug treatment shall I collect both adherent and floating dead cells, perform cell lysis and do WB or just collect adherent cells discard floating cells to proceed further.
Let me know your thoughts.
Thank you,
Anita
Hello Anita. It would be good to also collect the suspension, as your target protein might be released into the suspension. On your blot, you should also detect actin in the suspension to see the presence of dead cells.
I opine to collect both dead and live cells separately with or without drug treatment. Do not discard the media. Concentrate the media by freeze drying. Now you have three different preparations. You mentioned that you are testing expression of certain protein of interest. Giemsa staining will differentiate the dead cells from the live one. Challenge three preparations to detect your protein of interest. You can do it as per your methods. By dot blot or western blot comparing with cells without treatment.
Hi Anita. I have previously done WB with drug treatment in mammalian cultured cell lines as well, and I found not including the dead-cell suspension skewed my desired expression pattern (especially so if I was looking for apoptotic markers). So I'd suggest to include all of it.
Thank you so much Nurul, Mohammad and Arindam for your feedback. Very useful info. I have collected both the cells. Fingers crossed!
I guess what you need to be certain of initially is that the floating cells (whether they are dead or just detached) are not arising from the way the cells have been cultured rather than the drug treatment itself.
Good comment, Ian. That's why she needs to collect detached cells (and the medium) from the control dishes as well. Good luck, Anita!
Very good point Ian! Also Irina thank you for your feedback. Yes control cells don't have as many floating cells so its definitely the treatment. Moving ahead with collecting all of them and followed by western analysis.
Regards,
Anita
Thank you, Baydaa. I appreciate your opinion. Good luck in your research!
I agree with Ian. Best to first determine if the cells are really dead or just detached and then proceed further. Will save you a lot of trouble later. Best of Luck !
- Badges
- Science topic