Clone mpc 11

RRMS patients treated with natalizumab (n = 9) and healthy donors (n = 11) were recruited and PBMCs were isolated by density gradient centrifugation using Ficoll-Paque plus (GE Healthcare) and stored in liquid nitrogen until use. Upon thawing, cells were washed, counted, and resuspended in RPMI medium supplemented with 20% FCS, L-glutamine, and penicillin-streptomycin. Cells were plated at 2 × 10⁶ cells/mL and stimulated with 40 μg plate-bound TDB, 50 ng soluble LPS, or wells were treated with isopropanol prior to air drying as negative controls. Cells were cultured for 48 hours and treated with GolgiPlug (BD Biosciences) 5 hours prior to termination of the experiment. Cells were stained for MCL (clone 9B9, catalog 360204) and isotype (clone MPC-11, catalog 400314) expression prior to in vitro stimulations (BioLegend). Upon stimulation, cells were stained with LIVE/DEAD stain (Thermo Fisher Scientific, catalog L34976), CD14 (clone HCD14, catalog 325608), IL-8 (clone E8N1, catalog 511406), IL-6 (clone MQ2-13A5, catalog 501114), TNF (clone Mab11, catalog 502926), or isotype for cytokines (clone MOPC-21, catalog 400108) (BioLegend).

Cells were harvested using 500 U/mL accutase in PBS (Sigma-Aldrich). Cells were centrifuged and resuspended in a staining buffer consisting of PBS with 5% FBS, 1 mM EDTA (Sigma-Aldrich), 10 mM HEPES (Sigma-Aldrich). Then, 1 × 10⁵ cells per sample were stained with anti-human ABCB1 (348606, clone UIC2, 10 µg/mL; Biolegend, San Diego, CA, USA), anti-human ABCG2 (332008, clone 5D3, 5 µg/mL; Biolegend) or corresponding isotype controls (IgG2a: 400214, clone MOPC-173, IgG2b: 400314, clone MPC-11; Biolegend) conjugated with phycoerythrin (PE). After incubation for 30 min at 4 °C in the dark, the cells were centrifuged and then resuspended in a staining buffer containing 0.5 µg/mL 7-aminoactinomycin (Biolegend) for live/dead cell discrimination. The measurement was performed on a BD Celesta flow cytometer (BD Biosciences, Heidelberg, Germany) with excitation optics containing a violet (405 nm), blue (488 nm) and yellow-green (561 nm) laser. Flow cytometry data were analyzed using FlowJo software (BD Biosciences).