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Detect apoptosis by FACS - (Mar/02/2010 )

Hi,

My stable cell line (after stable transfection of my gene ) grows slower than my parental cell line. Furthermore, transient transfection of my gene of interest results in a cleaved PARP band. I'm assuming it could be playing a role in apoptosis.

I'd like to detect apptosis by detection of sub-diploid population in cell cycle by flow cytoemtry. E.g. how many cells in G1 and how many cells in S phase of cell cycle.

Do to this, I first nee dto transiently transfect my cell with my gene of interest. But the protocol requires 100000 (10 power 6) cells which is not feasible to transfect and also costly. I usually transfect 150000 cells. I can transfect at most 450,000 cells. Has anyone used about half a milion cells, instead of 1 million of cells required for flow cytometry?

-SF_HK-

SF_HK on Mar 3 2010, 09:24 AM said:

Hi,

My stable cell line (after stable transfection of my gene ) grows slower than my parental cell line. Furthermore, transient transfection of my gene of interest results in a cleaved PARP band. I'm assuming it could be playing a role in apoptosis.

I'd like to detect apptosis by detection of sub-diploid population in cell cycle by flow cytoemtry. E.g. how many cells in G1 and how many cells in S phase of cell cycle.

Do to this, I first nee dto transiently transfect my cell with my gene of interest. But the protocol requires 100000 (10 power 6) cells which is not feasible to transfect and also costly. I usually transfect 150000 cells. I can transfect at most 450,000 cells. Has anyone used about half a milion cells, instead of 1 million of cells required for flow cytometry?

I think the total amount of cells you have is not really important as long as you have enough. This depends on flow rate, concentration of cells per mL, and the amount of cells you want to analyse (10,000 or 20,000 are commonly used). This should have not impact on the measurement. If you use less cells, just resuspend them in a lesser volume of FACS buffer or whatever you use before measurement, so the poor cytometer does not have to work for 10 minutes or more. 100,000 cells should be fine.

Regardless of that, why is it not efficient to transfect 1,000,000 cells? Which method do you use for transfection? Most of the simple transfection methods, such as polymeric or lipid transfection reagents, can be used for high amounts of cells without using to much of the expensive materials required. I transfected about 10,000,000 cells in a total volume of ~ 15 mL using 20 ÁL of a polymeric transfection reagent without any problems.

-Aquifex-