Horizon fixable viability stain 510 dye

Mononuclear cell suspensions from decidual tissues were stained with the BD Horizon Fixable Viability Stain 510 dye (BD Biosciences) prior to immunophenotyping. Mononuclear cell suspensions were then washed with FACS staining buffer (CAT#554656; BD Biosciences) and incubated with 20µl of human FcR Blocking Reagent (CAT#130-059-901; Miltenyi Biotec) in 80µl of FACS staining buffer (BD Biosciences) for 10 min at 4°C. The cells were incubated with extracellular fluorochrome-conjugated anti-human monoclonal antibodies for 30 min at 4°C in the dark (Supplementary Table 1). After extracellular staining, the cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) prior to staining with intracellular and intranuclear antibodies (Supplementary Table 1). Stained cells were washed and re-suspended in 0.5 mL of FACS staining buffer and acquired using an LSRFortessa flow cytometer and FACSDiva 6.0 software (BD Biosciences). Data was analyzed using FlowJo software version 10 (TreeStar, Ashland, OR, USA).

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.

Amniotic fluid samples (0.5–1 mL) were centrifuged at 300 x g for 5 minutes at room temperature. The resulting amniotic fluid pellet was resuspended in 1 mL of 1X phosphate-buffered saline (PBS) (Life Technologies, Grand Island, NY, USA) and stained with BD Horizon Fixable Viability Stain 510 dye (BD Biosciences, San Jose, CA, USA). Cells were washed in 1X PBS and incubated with 20 μL of human FcR blocking reagent (Miltenyi Biotec, San Diego, CA, USA) in 80 μL of stain buffer (BD Biosciences) for 10 minutes at 4°C. Next, cells were incubated with extracellular fluorochrome-conjugated anti-human monoclonal antibodies for 30 minutes at 4°C in the dark (Supplementary Table 1). Stained cells were then washed with 1X PBS, resuspended in 0.5 mL of stain buffer, and acquired using the BD LSR II or LSRFortessa Flow Cytometer (BD Bioscience) and BD FACSDiva 6.0 software (BD Bioscience). The analysis was performed, and the figures were generated using the FlowJo version 10 software (FlowJo, Ashland, OR, USA). The absolute number of cells was determined using CountBright absolute counting beads (Molecular Probes, Eugene, OR, USA).