Lsr 2 or lsrfortessa flow cytometer

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).

Amniotic fluid samples (0.5‐1 mL) were centrifuged at 300 × 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).