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
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.
I didn't stop the trpsin reaction this time, only washed the cells with PBS. I think that's the reason for this.
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 on Fri Mar 28 15:40:48 2014 said:
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.