Il 8 clone e8n1

Cells were surface stained with anti-human antibodies (Abs) to CD11b (clone ICRF44), CD14 (clone TuK4), CD31 (clone M89D3), CD45 (clone 2D1), CD163 (clone GHI/61), IL-6 (clone MQ2-39C3), TNF-α (clone MAb11), vimentin (clone RV202) (BD Pharmingen, San Diego, CA), IL-8 (clone E8N1), CD326 (EpCAM, clone 9C4) (Biolegend, San Diego, CA), and CD3 (clone UCHT1, Beckman-Coulter, Miami, FL). Antibodies conjugated to the following fluorochromes were used in these studies: Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), Peridinin chlorophyll protein (PerCP)-Cy5.5, PE-Cy7, Energy Coupled Dye or PE-Texas-Red conjugate (ECD), Pacific Blue, Brilliant Violet (BV) 570, BV605, BV650, Quantum dot (QD) 800, Alexa 647, allophycocyanin (APC)-Alexa 700 and APC-H7.

Cells were surface stained with anti-human monoclonal antibodies (mAbs) to CD11b (clone ICRF44), CD11c (clone B-ly6), CD14 (clone TuK4), CD45 (clone 2D1), CD66c (clone B6.2/CD66), CD163 (clone GHI/61), IL-6 (clone MQ2-39C3), TNF-α (clone MAb11), (BD Pharmingen, San Diego, CA), CD19 (clone SJ25-C1), CCL3 (clone CR3M)(Invitrogen, Carlsbad, CA), IL-8 (clone E8N1), and CD326 (EpCAM, clone 9C4) (Biolegend, San Diego, CA). These mAbs were directly conjugated to the following fluorochromes: Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), Peridinin chlorophyll protein (PerCP)-Cy5.5, PE-Cy7, Energy Coupled Dye PE-Texas-Red conjugate (ECD), Pacific Blue, Brilliant Violet (BV) 570, BV605, BV650, Quantum dot (QD) 800, Alexa 647, allophycocyanin (APC)-Alexa 700, or APC-H7.

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).