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preparation of cells for FACS analysis 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 do you inactivate your Trypsin? I usually use a solution of 5% FCS with 3mM EDTA in PBS for resuspension of cells, this inactivates Trypsin and prevents clogging of the cells.

-Rsm-

I didn't stop the trpsin reaction this time, only washed the cells with PBS. I think that's the reason for this. 

-Push-

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

-Push-

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

 

I also struggle against very variable transfection rates. I make sure that my stock culture is 80-90% confluence prior to harvesting and plating (and plate the cells at the same density every time) and then I transfect (Lipofectamine 2000) 16.5-18 hours after plating. Then I remove the medium and trypsinise (using TrypLE) 24 hours later to reduce the confluence. I'm not sure what would happen to my cells (primary chicken embryo fibroblasts) if I let them sit there at 100% confluence but I think they might differentiate and form a sheet before delaminating from the culture surface.

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