Cd86 fitc

γδ T cells were phenotyped after 48-hour co-culture using the following monoclonal antibodies (mAbs): γδ TCR-FITC (Miltenyi), CD86-FITC, CD80-PE, PD-1-PE, NKG2D-PE, CD16-PB, BTLA-BV421, γδ TCR-APC (Miltenyi), NKp30-AF647, and CD3-APC-H7. Unless specified otherwise, all mAbs were purchased from BD (Erembodegem, Belgium). Live/Dead^® Fixable Aqua Stain (Invitrogen, Merelbeke, Belgium) was used to exclude dead cells from analysis. Corresponding species- and isotype-matched antibodies were used as controls. The corresponding gating strategy can be retrieved from Figure S1 in Supplementary Material.

Next, 24 h after LPS stimulation, BMDCs were detached from the plates with DPBS 1× (Gibco, MA, USA) + 0.5mM EDTA (Thermo Fisher Scientific, MA, USA). Cells were then washed with DPBS 1× + 0.5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) and labelled with CD11b VioBrightFITC (Miltenyi Biotec, Bergisch Gladbach, Germany), MHCII PE (Miltenyi Biotec, Bergisch Gladbach, Germany), CD11c PECy5 (Miltenyi Biotec, Bergisch Gladbach, Germany), F4/80 APC (Miltenyi Biotec, Bergisch Gladbach, Germany), CD80 FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), CD86 FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), and 7-AAD PECy5 (Miltenyi Biotec, Bergisch Gladbach, Germany). Flow Cytometer data analysis was performed using NAVIOS software (Beckman Coulter, Brea, CA, USA), with at least three experiments performed. Flow cytometer analysis was performed using Kaluza Software 1.5 (Beckman Coulter, Brea, CA, USA).