Cd15 bv605 clone w6d3

Mononuclear cell suspensions from decidual tissues were stained with BD Horizon Fixable Viability Stain 510 dye (BD Biosciences) prior to incubation with extracellular mAbs. Mononuclear cell suspensions were then washed with staining buffer (Cat No. 554656; BD Biosciences) and centrifuged. Cell pellets were incubated for 10 min with FcR Blocking Reagent (Cat No. 130-059-901; Miltenyi Biotec). Next, mononuclear cell suspensions were incubated with the following fluorochrome-conjugated anti-human mAbs: CD14-BUV395 (clone MφP9; BD Biosciences), CD15-BV605 (clone W6D3; BD Biosciences), CD3-BV650 (clone OKT3; BD Biosciences), CD19-BUV737 (clone SJ25C1; BD Biosciences), CD56-PE-Cy7 (clone NCAM16.2; BD Biosciences), CD69-Alexa Fluor 700 (clone FN50; BD Biosciences) and Vα24Jα18TCR-PE (clone 6B11; eBioscience; San Diego, CA, USA) for 30 min at 4 ºC in the dark. Finally, mononuclear cell suspensions were washed and re-suspended in 0.5ml of staining buffer and acquired using the BD LSRFortessa flow cytometer and FACSDiva 6.0 software. Leukocyte subsets were gated within the viability gate, and activated iNKT-like cells were identified as CD15−CD14−CD19−CD3+CD56+CD69+ or CD3+Vα24Jα18TCR+CD69+ cells. The analysis was performed, and the figures generated using FlowJo Software version 10.