Hi Punita,

here you'll find protocols for transfecting lots of different cell types:

https://www.sigmaaldrich.com/technical-documents/protocols/biology/xtg-cell-protocols.html

Here is a detailed protocol for transfecting cells and consecutive FRET assay. Article FRET and BRET-based biosensors in live cell compound screens

Hope that helps

Hi Punita,

Well, when I did transfection in 96 wells plate, first I seeded each well with 1x104 cells and filled each well with 200 uL of DMEM. Then I put the plate in an incubator for 24 hours. After that, I withdrawn the DMEM and washed each well with PBS for 2 times. This because in my case, the cells that I used in a 96 well grown 60-65% (of confluence) in 24 hours when I seeded the well with 1.000 cells in each well. So you will need to do a growth curve to determine how much cells and how much time do you will need to achieve 60-65% in confluence per well. After that, you will can do the transfection protocol normally.

I hope that it helps you.

Pedro

FIRST DAY: seed cells. the optimum number of seeding cells per well vary on the cell type, as different cells have different diameters.

I recommend to thy seeding different concentrations: usually:

10x105 20x105 30x105 40x105 50x105 60x105 70x105 80x105.

Be careful to assure equal spreading of the cells. Avoid making bubbles, as in this case the cells wont attach to the surface where the bubble is!

SECOND day: check the cells on the microscope. Carefully select the concentration of cells that corresponds to 60% of confuency. Only these wells will be used for the transfection!

Then, suck off the media and put 100 uL DMEM.

Prepare the reagent transfection mixture:

Recommended Reagent:DNA ratio for most of the cells is 3.0 :1; it means:

in a eppendorf tube add 2.2 µg of DNA, 6.6 µl of FuGENE® HD reagent and OptiMEM till 103 µl. MIX THOROUGHLY! Then incubate the mixture for 15 minutes; then from this mixture, add 10 µL per well.

THIRD DAY: suck off media and put a fresh one.

FOURTH DAY (42 h after the transfection): image!