You may have added too much of the transfectamine you did not change media on time

Hi Samna

Perhaps the stable transfection did occur in very few cells and hence you observed a massive cell death as most cells could not survive under the selection pressure of an antibiotic. In stable cell line generation, as you are trying to clone a foreign sequence into genomic DNA, the chances of cells acquiring such foreign DNA would be quite low. The massive cell death that you observed is therefore not surprising and even if you have very few cells that are surviving you should continue to grow them.

Did you passage them further after your transfection when they were fully confluent? I am assuming that since at the time of your transfection the cells were already 80% confluent so in one or two days' time, they will be fully confluent and may start to detach after that leading to cell death. To rule out this possibility, did they show any morphological changes or stress signals indicative of cell death (which is irrespective of whether or not the transfection occurred)

Also since you used different cells this time these cells might not be suitable for your experiment in the first place (Hypothetically speaking)

What is the cell passage number used for transfection as it should be low when performing transfection-related experiments?

The following link states that lipofectamine messenger MAX may be more suitable than lipo2000 for neuronal cell transfection

https://www.thermofisher.com/in/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html

Also, you can see cell-specific transfection protocol here https://www.thermofisher.com/in/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html

Hi Bright Kwaku Anyomi

My DNA: Lipofectamine ratio was 1:2. I did change the media the very next day (Friday), waited over the weekend and when I checked it out on Monday almost 95% of the cells were dead. I immediately decreased the antibiotic concentration and changed the@ media, but does not seem to help.

Hi Vikrant Mehta

Thanks for your time and suggestions.

I did not passage the cell as they were starting to degrade on the very third day. I generally wait until the seventh day for individual cells to form pure colonies and then subculture them separately.

There are still few cells adhered to the plate, but clearly, they seem to be stressed and undergoing degradation.

I have set up another plate for transfection today with decreased concentration of the antibiotic. I will let you know the outcome.