I want to test transfection efficiency using a plasmid encoding GFP (green fluorescence protein). We do not have a fluorescence microscope, but we do have a plate reader. I am planning to grow the HCC827 cells in 6 well plates and transfect them. After 48 hours I plan to scrape cells (or trypsinize them) to detach them from the plates, collect them in PBS and read them in a plate reader. I will have the cells alone and cells with transfection reagent (no DNA) as negative controls for auto fluorescence correction. Will I be able to read GFP fluorescence using a plate reader following this method? Please let me know if you have any suggestions or anticipated problems. The signal can be very low and I may have to lyse cells. Thank you!
Hi!
Fluorescent microscope is the best equipment for this research, but if you have a plate reader, you can do cell lysis using any lysis buffer (check their ability to decrease signal) and put the solution into plate reader. Use any "blue" light (dependently of reader filters set) for activation of GFP is solution. Detection in "green" area of specter. Lysis buffer with cell without GFP as control.
Regards,
Tatiana
Dear Paromita,
Transfection efficiency refers to the number of transfected cells, relative to the total number of cells. You need either a microscope or a cell sorter to determine this.
What you will get from the plate reader is the amount of GFP in your cell culture. This depends on both, the transfection rate AND the expression level. That may also be useful for your application, but you need to be aware that is is not synonymous to the transfection efficiency. In any case, if you want to know the GFP levels in the culture, this is usually tested by western blotting using GFP-specific antibodies. You would then also avoid the autofluorescence problem, but you still need non-transfected cells as a control.
Dear colleagues!
Let me continue this discussion. I used ICC for GFP transfection effectiveness (fluorescence microscopy). Some cells didn't show any GFP fluorescence but GFP specific antibodies recognized GFP well. Returning to transfecting effectiveness checking, You can use any other protein for reaching this goal. In my opinion, WB should to be more effective for transfection effectiveness checking but requires more steps, reagents and hours.
Regards,
Tatiana
Thank you all for your suggestions. I actually tried transfecting my cells using different conditions in a 96-well plate. But the plate reader could not detect any signal. My take is since the amount of cells were less, the signal was too low. Since I tried various conditions, its unlikely that none worked. I am planning to use a 6-well plate, lyse the cells post transfection and detect using the plate reader. In case there's still no signal, I will do a Western blot.
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