2.5. Radioligand binding experiments

Details of the radioligand binding experiments are in the supplementary methods. In brief, brain tissue was obtained from 4 HABs and 4 low affinity binders (LABs) from the UK Multiple Sclerosis Tissue Bank at Imperial College and stored at −80°C until use. Binding affinity status was determined previously by radioligand binding experiments using [3H]PK11195 and unlabelled PBR28. ³⁴ Aliquots of membrane suspension were incubated with [3H]PK11195 (0.3 nM) and 1 of 8 concentrations of etifoxine ranging from 150 nM to 30 μM. The assay was terminated via filtration through Whatman GF/B filters. Scintillation fluid (4 mL/vial, Perkin Elmer Ultima‐Gold MV) was added and vials counted on a Perkin Elmer Tricarb 2900 liquid scintillation counter. A dissociation constant for [3H]PK11195 of 29.25 nM⁶ was used to generate the inhibition constant (Ki) for etifoxine according to the Cheng and Prusoff Equation ¹² using GraphPad Prism 8.1 software (GraphPad Software Inc, USA).