Single use red plate with inserts

Plasma free fraction (f_P) was measured using rapid equilibrium dialysis (RED). Plasma samples were isolated by centrifugation from whole blood withdrawn from each participant before radiotracer injection. A 100 μl sample of plasma was spiked with 1 μl of [³H]DPA-713 (83 Ci mmol⁻¹; Quotient Bioresearch) and added into the sample chamber of Single-Use RED Plate with Inserts (Thermo Scientific, Rockford, IL, USA) with an 8 K molecular-weight cutoff. Then, 300 μl of PBS was added to the buffer chamber and the plate was incubated on an Incubating Microplate Shaker (Fisher Scientific, Waltham, MA, USA) at 37 °C for 4 h. Ten microliters of plasma and 30 μl of PBS were transferred to scintillation vials and mixed with 10 ml of Bio-Safe II (Research Products International, Mount Prospect, IL, USA) counting fluid. All the samples were measured in triplicate. The radioactivity was measured using an LS 6500 Multi-Purpose Scintillation Counter (Beckman Coulter, Pasadena, CA, USA) to obtain the radioactivity in the plasma (C_P) and buffer (C_U). The free, unbound fraction (f_P) was calculated as: f_P=C_U/C_P × 100 (%).