Hello Sar Bdt

1. Should I remove the culture media or change it or decrease it before transfection? Would that have an effect?

You should remove the culture media and replace with growth media without serum. Add the diluted complex to each well and mix gently by rocking the plate.

2. Should I remove the transfection media after 6 hours of transfection or can it stay for the next day in the incubator?

The optimal time interval will depend on the cell type. Some cells are resistant to taking up components from the environment and are therefore difficult to transfect. They will need longer time to undergo transfection. But for A549 cells, you may replace with complete growth media within 4-24 hours post-transfection.

I have attached the protocol of transfection of A549 cells using lipofectamine 3000 reagent in a 24-well plate.

https://www.thermofisher.com/in/en/home/references/protocols/cell-culture/transfection-protocol/a549-cells-protocol.html

Best.

Hi Sar Bdt

I had a tough time transfecting A549 cells using Lipo-3000. I used different concentrations of plasmid mixed with Lipofectamine (1:3 w/v), the mixture being in Opti-MEM for 6-18 hrs (as recommended by the manufacturer) and subsequently changed to growth medium (supplemented with 10% FBS) for another 24 hrs.

For me the transfection efficiency was very poor as compared to hRPE-1 cells (might be a problem with the passages of the A549 I was using).

But would be happy to know if you get more or less of a standard number of transfected cells.