I transfected Hela cells with GFP-tagged protein in corning-3603 black clear bottom 96-well plate and tried to use biotek cy3 microplate reader to detect GFP flurorescence intensity with excitation filters 485 BP and emission filter 528 BP. However, I found the background flurorescence in non-transfected wells was even higher than which transfected with GFP, so how can i reduce this background flurorescence? Or any other plate suitable for my assay? Thank you very much!
I would suggest different excitation and emission filters. Use 395 nm wave length for excitation and 509 nm for emission because these are peak areas for GFP. Other stuff like plate reader and plates are OK
How is your microplate reader working? Where does it measure the emission, above or under the plate?
We use black bottom plates and not clear bottom.
Also, you can check it the well bottoms are flat or curved. That can affect also.
Hi! I usually set excitation to 484nm and emission at 510nm and it works well for me. Excitation and emission bandwidths are at 10nm. Parameters on my plate reader also have Integration Time (20us), Lag Time (0us) and Settle Time (10us).
Did you change the medium in wells to PBS just before measuring? It creates high background otherwise
May You know how THIN/film polystyrene bottom of fluorescence plate compares with cycloolefin film? I link two kinds of plates for example.
https://www.thermofisher.com/order/catalog/product/165305?SID=srch-srp-165305#/165305?SID=srch-srp-165305
https://shop.gbo.com/en/spain/products/bioscience/cell-culture-products/microscopy/en-screenstar-microplates/655866.html
Have a nice day! Stay healthy!
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