Pd l2 clone mih18

The following mAb were used for flow cytometry analysis: CD56 (clone NCAM1), CD16 (clone 3G8) CD3 (clone SK3), CD11b (clone ICRF44), CD14 (clone MOP9) PD1 (clone EH12.1), PD-L1 (clone MIH1), PD-L2 (clone MIH18), CD138 (clone MI15) MICA/B (clone 6D4), CD155 (clone TX24), NKG2A (clone 131,411), NKp44 (clone p44-8), TIM3 (clone 7D3), TIGIT (clone 741,182), DNAM1 (clone DX11) from BD Biosciences; HLA-ABC (clone W6/32), CD38 (clone HIT2), NKp30 (clone p30-15), NKp46 (9E2/NKp46), LAG3 (clone 11C3C65), NKG2D (clone 1D11), CD112 (clone TX31) from BioLegend; CD158a/h/g (clone HP-MA4), CD158e (clone DX9) from Thermofisher, CD158B (clone GL183) from Invitrogen, ULBP256 (clone 165,903) from R&D. All antibodies were titrated prior to usage. Briefly, cells were collected and washed once in PBS. Cells were stained with Aqua live dead cell staining (ThermoFisher) for 20 min at 4 °C, in the dark. Cells were washed once with PBS, containing 2% FBS. Surface staining was performed for 25 min at 4 °C, in the dark. Cells were washed with PBS, containing 2% FBS, centrifuged and fixed with 1% paraformaldehyde for 10 min. Acquisition was performed the following day with Beckman Coulter Cytoflex flow cytometers. Analysis was performed with FlowJo analysis software version 10. Gates were — unless otherwise specified – placed based on the unstained control or FMO (fluorescence minus one).

After thawing, cells were stained with directly labeled antibodies recognizing CD33 (clone P67.6; PE-conjugated), CD3 (clone SK7; PerCP-conjugated), CD45 (clone 2D1; FITC-conjugated), PD-L1 (clone MIH1, PE-conjugated), PD-L2 (clone MIH18, BV650-conjugated), CD86 (clone 2331, APC-conjugated), CD34 (clone 8G12; APC-conjugated; all from BD Biosciences) and CD80 (clone 2D10, PE-Cy7-conjugated; from BioLegend). To identify nonviable cells, samples were stained with DAPI. At least 10 000 events were acquired on an LSRII flow cytometer (BD Biosciences), and DAPI^- cells were analyzed using FlowJo.