“Negative control” is a treatment that by definition is expected not to have any effect (neither positive effect, nor negative effect). “Positive control” is treatment with a well-known chemical that is known to produce the expected effect with the assay that you are studying. Application of an antagonist is not a negative control in your case. “Negative control” is condition that should be treated with the same solutions or buffers as your “treatment” condition, with the only difference that instead of the chemical that you investigate you should add just the solvent that was used to dissolve you chemical in the respective final concentration that you have in the “experimental treatment” condition. For example if your chemical is dissolved in DMSO – than the correct negative control will be to add to the medium/buffer just DMSO in the same final concentration that you reach with your “treatment” condition. One of the reasons of using such negative control is to verify that the solvent is having no effect in your assay. Note that among all treatment conditions (“negative control”, “positive control”, “experimental treatment you are investigating”) the volumes and the composition of the treatments that you are doing should be uniform: always treat with the same volume of medium or buffer, always containing the same concentration of the used solvent (e.g., DMSO). The only difference should be the presence or absence of the defined compound-treatments (agonist, antagonist, the chemical for the experimental investigation etc.).

I think one of your negative controls will be the cell line treated with the solution or buffer which you use to dilute the agonist, then follow the same procedure to isolate protein. As you mentioned nonhalucinogenic will be the best one for the negative control.

As for the positive, you could get the extract from the animal origin when the agonist shows activity.

I agree with Jingjing's answer for the negative control. Yes DOI should act as a positive control there.

“Negative control” is a treatment that by definition is expected not to have any effect (neither positive effect, nor negative effect). “Positive control” is treatment with a well-known chemical that is known to produce the expected effect with the assay that you are studying. Application of an antagonist is not a negative control in your case. “Negative control” is condition that should be treated with the same solutions or buffers as your “treatment” condition, with the only difference that instead of the chemical that you investigate you should add just the solvent that was used to dissolve you chemical in the respective final concentration that you have in the “experimental treatment” condition. For example if your chemical is dissolved in DMSO – than the correct negative control will be to add to the medium/buffer just DMSO in the same final concentration that you reach with your “treatment” condition. One of the reasons of using such negative control is to verify that the solvent is having no effect in your assay. Note that among all treatment conditions (“negative control”, “positive control”, “experimental treatment you are investigating”) the volumes and the composition of the treatments that you are doing should be uniform: always treat with the same volume of medium or buffer, always containing the same concentration of the used solvent (e.g., DMSO). The only difference should be the presence or absence of the defined compound-treatments (agonist, antagonist, the chemical for the experimental investigation etc.).

Thanks all for your kind feedback. Hope these will help a lot. Yes I agree with all of you. So now I may use DOI as positive control, my compound (agonist of 5-HT2A receptor), crude extract, selective antagonist (Ketanserin, as it inhibits both PLC-IP accumulation and PLA2-AA release), negative control as you mentioned sequentially in my experiment on 5-HT2A neuronal cell line for in vitro activity.

But one thing still I want to know how will I fix the dose for positive control, selective antagonist as well as the crude extract (from animal origin). I know the dose of my compound which is agonist (it will be from nonhallucinogenic to fair hallucinogenic). Should the dose of crude extract be kept same as like as my compound (here notable that the compound of interest is in the crude extract along with many compounds)?

What factors need to be considered to select the dose of positive control, selective antagonist and also for crude extract?

It will be much helpful if any have the answer of these.

Thanks again.