I want to work with the 5-HT2A neuronal cell line with the agonist and a crude extract from animal origin. My intention is to observe whether they upregulate or downregulate the receptor. My assumption is that both should upregulate the receptor cell line. The agonist is hallucinogenic but I will use non-halucinogenic to fair hallucinogenic dose and the crude extract also has some mind altering activity. The signalling pathway of the agonist is mainly Gq/11 PLC-IP3 and also mild PLA2-AA .
So what are the factors here to select the positive control and negative control? Should I use DOI (as it activates both Gq/11 PLC-IP3 and PLA2-AA pathway) as the positive control here? If I use a selective antagonist of 5-HT2A receptor activating Gq/11 PLC-IP3 or both Gq/11 PLC-IP3 and PLA2-AA pathway then will it be a negative control?
In general, controls are used to (1) ensure your experiment is working (2) eliminate the possibility that the result you see is not from an unintended source. So the more controls you have, the better. The amount and depth of controls needed also depends on the question you are asking (in this case: Does the presence of the agonist increase receptor expression?).
Positive control: In this case, the specificity of the agonist (DOI) for the receptor is key, rather than using it because it activates some relevant downstream pathways (Remember, your initial question asks whether receptor expression increases in the presence of the agonist, not, what is the pathway through which receptor expression occurs). So once you are happy about the specificity of the agonist, its a good positive control.
Negative control: In choosing the negative control using treatment groups (a) with a selective antagonist (b) with a broad specificity antagonist would be helpful. You should also clarify whether you want to block initial activation of your receptor (a receptor antagonist) or inhibit the downstream signalling pathway (i.e. inhibit the signalling pathway). The first option is probably more useful for you in this case but the latter is useful if you want to study the mechanism. Again antagonist specificity is key. Finally, make sure to use the antagonist in the presence of the agonist (i.e. the same treatment group) as well as the antagonist on its own (to show it has no effect).
Thanks Omar for your nice feedback. One thing I want to know, why the antagonist should be used in the presence of agonist. I have also seen it in some publications but could not able to find the correct answer. Could you please help me in this regard?
Another thing, how the Ki value helps in dose calculation for agonist or antagonist used in the experiment. Say for example, in one compound the Ki value is 0.034 nm, so how the conc. would be 1 nm (seen in one article)?
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