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Dead cells after transfection - (Mar/13/2014 )

Hello,

 

I am PhD student and I did a transfection experiment for three tumour cells lines. I found all my cells were dead. Please help me.My experiment was as below.

 

I tried to optimize the transfection with turbofectin 8 (origene) and lipofectamin (Invitrogen) for three cell lines. I did  according to the manufactures guidelines. Cells were live and were in a more than 90% confluence before I tripsynized them after 48 hrs for FACS analysis. Cells were in EMEM and DMEM (Complete medium). As I was optimizing the protocol, used the pmax-GFP plasmid and I washed the cells in PBS. I centrifuged the cells to remove PBS and found cells in clumps. For FACS, resuspended the cells in PBS and didn't do cell fixation. 

 

Please help me.

 

Thank you

-Push-

How did you determine the cells were dead?  Clumping is not an indication of death.

-bob1-

I analysed the cells by flowcytometry using a viability dye (Viodye- e780) and found out 99 % of dead cells

-Push-

were your cells alive before you centrifuged?

 

if so then you may want to try more gentle conditions when centrifuging.

 

if not then you should check viability after each step in your procedure to find out which one is the problem (or, at least, from trypsinization onward).

-mdfenko-

How did you purify the DNA that you are transfecting? What method did you use?

 

I am asking this as I am concern about endotoxin contamination coming from your DNA.

 

Also might your DNA be in a tris buffer? Tris is toxic to cells.

-perneseblue-

Hello All,

 

I forgot to stop the reaction by adding medium with serum,instead I washed the cells after the trypsinization. Maybe that could be the problem.This time I used a commercially purchased plasmid pmax-GFP to optimize the protocol with Turbofectin 8.

-Push-

Hello All,

 

I forgot to stop the reaction by adding medium with serum,instead I washed the cells after the trypsinization. Maybe that could be the problem.This time I used a commercially purchased plasmid pmax-GFP to optimize the protocol with Turbofectin 8.

Have you solved the problem yet?

-Chen Wang-

It looks like they did solve it - they didn't neutralize the trypsin...

-bob1-

Hello All,

 

I have repeated the the transfection with cancer cells of epithelial origin (MCF -7, MDA-MB-231, BT-20) using Turbofectin 8 and with pRFP-C-RS and pmax-GFP but ended with a very low transfection efficiency. My cells were live but with no transfection.

 

Hope you all could help me

 

thank you

-Push-