Good day everybody,
i am currently trying to transfect ARPE-19 cells with a GFP Plasmid (pEGFP-N1-FLAG; Plasmid #60360).
I am using Viafect which works really well (transfection rate of over 95 percent). However 24h-48h hours post transfection my cells all die. I dont understand why this is so, since it is an only GFP Plasmid and should not lead to cell death.
I also tested the transfection reagent viafect alone without any Plasmid and the MTS/ KV revealed that it is not harmful for the cells.
I also tried different DNA: Transfection reagent-ratio´s which lead to cell death depending on the amount of cells transfected. If less cells were transfected, less cells died, so i think it is related to the process of the cells expressing the Plasmid ?
Is it possible that the N1-FLAG on the plasmid is harmful for the cells ?
If anybody got an idea why this is so, what i could do in order to solve this Problem i am very thankful for your help,
Best
Peter
(The first picture is the 24h post Transfection, the second 48h and the third an untreated control)
ARPE-19 cells don't mind to exist as a cellular monolayer for months at a time, they actually differentiate over time. Many plasmids induce the Caspase-3/8 dependent apoptosis this is quite a common occurrence as cells react to the presence of the foreign genetic material or foreign protein it might have to do with a rather fast N1 promoter that you have (at 24 cells are already have a strong GFP fluorescence). This problem is solved by Z-VAD-FMK to block caspases and you will keep your 95% efficiency. Although I was successful in transfecting ARPE-19 cells with EF1a-GFP plasmids and other large constructs without issues, EF1a is a more slow promoter and you get noticeable expression only after 48 hours and not 24.
Hi, Cells were over confluent. Transfect cells when they are about 60% confluent. Over confluency can also kill cells.
ARPE-19 cells don't mind to exist as a cellular monolayer for months at a time, they actually differentiate over time. Many plasmids induce the Caspase-3/8 dependent apoptosis this is quite a common occurrence as cells react to the presence of the foreign genetic material or foreign protein it might have to do with a rather fast N1 promoter that you have (at 24 cells are already have a strong GFP fluorescence). This problem is solved by Z-VAD-FMK to block caspases and you will keep your 95% efficiency. Although I was successful in transfecting ARPE-19 cells with EF1a-GFP plasmids and other large constructs without issues, EF1a is a more slow promoter and you get noticeable expression only after 48 hours and not 24.
You said you tested transfection reagent alone and it's not causing cell death. So must be something about your plasmid. As Adam Figarski said eGFP can be quite toxic. You're probably using too much plasmids and GFP expression is too high. I'd suggest titrate down the plasmid you use, or maybe remove and add fresh medium 8-12h after transfection
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