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Dead cells after transfection


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#1 Push

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Posted 13 March 2014 - 09:13 AM

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



#2 bob1

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Posted 13 March 2014 - 11:54 AM

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



#3 Push

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Posted 14 March 2014 - 12:24 AM

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



#4 mdfenko

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Posted 14 March 2014 - 03:53 AM

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).


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#5 perneseblue

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Posted 16 March 2014 - 08:32 PM

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.


May your PCR products be long, your protocols short and your boss on holiday

#6 Push

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Posted 17 March 2014 - 06:56 AM

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.



#7 Chen Wang

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Posted 24 March 2014 - 01:30 AM

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?



#8 bob1

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Posted 24 March 2014 - 11:37 AM

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



#9 Push

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Posted 28 March 2014 - 07:39 AM

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



#10 Olivia Zabel

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Posted 19 November 2014 - 12:51 AM

Hi,

 

for transfection of my tumor cell lines I use a new kind of transfection reagent which is called Viromer and was developed from Lipocalyx (www.lipocalyx.de).

 

Viromers are syntehtic polymers zero in charge and very gentle to cells. Due to their active endosome escape mechanism I get higher transfection efficiency compared to standard transfection reagents.

 

Pease find attached some data and inormation.

Attached Files


Edited by Olivia Zabel, 19 November 2014 - 12:51 AM.





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