4.5. Competitive Radio-Ligand AhR Binding Assay

Ligand binding assay was performed using cytosolic protein extracts from murine Hepa1c1c7 cells as described [37]. Briefly, aliquots of protein (2 mg/mL) were incubated at the room temperature for two (2) h with 2 nM [³H]-TCDD in the presence of DMSO (vehicle control), FICZ (positive control), 200 nM TCDF (non-specific binding) or increasing concentration of MICT. After the incubation, the hydroxyapatite slurry was added to the samples and the suspension was incubated on ice and washed three times with HEGT buffer. The hydroxyapatite pellet was re-suspended in scintillation cocktail, and radioactivity was determined in a liquid scintillation counter. The specific binding of [³H]-TCDD was determined by subtracting the radioactivity of non-specific reaction (TCDF) from total radioactivity. The values of IC₅₀ were calculated where appropriate.