How to measure inhibition of GFP expression with FACS?

07 July 2016 2 7K Report

I am trying to measure the GFP inhibition when transfected with siRNA targeting the GFP.

For that I am trying to use our FACS Diva.

I have the A549 cells stably transfected with GFP and I am trying to target the GFP with siRNA using transfection reagent.

I have did that and measured the decrease in the flurecence using a plate reader and it work as I noticed a decrease in the flurecence but when I used the FACS it didnt work.

Can you please advice of a correct way of doing that with FACS.

What I did is gating the untreated cells and I was expecting to see a shift in the peak of the count vs FITC back with the treated cells as an indicator for the GFP inhibition... is that correct? and is the use of FITC correct with GFP?

can anyone please advice.

Regards

Mohammad Obeid

Brian Lin

First thing, yes, FITC filters for GFP is fine.

Are you saying that after siRNA treatment, there was no change in the GFP signal at all? Or simply that with the gate you had, there was no difference in the percentage reported back for that gate?

If you are seeing a decrease in plate reader and by eye, it should definitely be detected by FACS, and seen as you described: a high GFP peak in control, and a shift in siRNA targeted cells back to baseline. It won't be 100%, but you should see some kind of difference.

I assume your GFP+ and negative populations were tested, to make sure your voltages on detectors are set correctly? As in, GFP+ around 3/4 max signal, and GFP- controls the bottom 1/4th?

I'm not super familiar with the FACS Diva, but if all the above is right, maybe check the filters in your machine? In our core, sometimes people will change the filters used for their fluor, and not change them back? I'd assume you'd see a difference in your FITC/GFP signal, but you never know...

Good luck!

Mohammad Ali Obeid

Dear Brian,

Thank you very much.

in the gate I had, there was no difference in the percentage reported back for that gate that's why I think there was no effect at all.

I didnt use negative control cells ( -ve GFP) as my cells are stabely transfected with GFP.

thanks

Mohammad

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