Hi all,
I am using the Fluo4-AM kit (ready to use, no washes needed) to investigate the activation of a given GPCR receptor. I am using HEK293T cells, co-expressing the GPCR together with Gq protein, and 48h, Fluo4-AM is loaded into the cells.
I am using a multi-plate reader equipped with an injector, that injects an agonist of said receptor. The signal is measured before and after the agonist injection, every second during 120s. I see NO difference in the signal.
Such an assay has been reported in the literature multiple times. I made sure that both Fluo4AM and the settings of the machine are correct by using a calcium ionophore (which reached saturation of the machine in a few seconds).
I have also been advised to keep the temperature constant, being at RT or 37C, which I did. No improvements there. I am also using published constructs that have been shown work.
Do you have any suggestions?
I would need to use a homogeneous assay, in a plate mode.
thank you in advance,
ARS
Hi,
Can you see the cells under an epifluorescence microscope to make sure you are loading them correctly. I would try with a test experiment to make sure the machine is OK.
If your ionophore is working then it's likely your assay is working. It could be your experimental drug does not induce your receptor.
Hi Ana Rita,
I agree with Jordan, if you can see the signal it means that technically it is working and if you don't see the differences before and after it possible means that the agonist is not working.
Which agonist are you using? Each cell type is different and the agonists are differently efficient according with the receptors expressed in the cells. First suggestion: try with a different agonist.
Second suggestion: add excess of Ca2+ on top of your cells together with a ionofore and see if in this case the signal increases. if yes, it means that technically is everything fine, you have just to better set up the agonist stimulation.
Good luck!
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