I have to perform fluorescence based assays in 96-well plate using HEK-293 cells. The experimental procedure involves transfection in cells and then treatment with drugs for testing drug efficacy. The problem I am facing is that before I finish experiment 80% of the cells gets detached from the surface. What can I do to resolve the issue?
I was using poly-L-lysine for transfected HEK293 cells and adhesion was good despite the washing steps in my procedure.
One possible setting would be the use of a matrix/coating for better adhesion of HEK cells to your plates. An alternative would be the use of better (prepared) plastic goods (96-well plates, e.g. from TPP). Using this stuff I get better results regarding HEK adhesion during experiments.
Andreas Eisenreich
Would matrix will effect the transfection process?
Hello Puneeta Bathla
In general coating is not needed for HEK cells. But I use Fibronectin or Gelatin, to coat the 96 well plates, I transfect HEK cells.
Transfcetion efficency was better in Forward transfection than reverse transfcetions.
FYI:
Regarding florescent read out:
https://ecatalog.corning.com/life-sciences/b2c/US/en/Microplates/Assay-Microplates/96-Well-Microplates/Falcon%C2%AE-96-well-Polystyrene-Microplates/p/353219
The imaging quality is far better with this plates than the regular 96 well plates.
Goodluck.
Best
Harsha
polykationic matrixes should effect the transfection. i experienced that.
Attila Lehotzky
Thank you very much for your feedback and I do defentely agree.
I had problems in the past with the reverse transfcetions, so I switched to forward tranfections.
Best
Harsha
I was using poly-L-lysine for transfected HEK293 cells and adhesion was good despite the washing steps in my procedure.
concerning your protocol, i would suggest first transfect your cells in a bigger vessel, than after a while, plate them onto 96 well plate coated poly-L-lysine.
- Badges
- Science method