Zombie red viability dye

Single cell suspensions of ear skin were prepared by incubating dermal side down in 500 uL 0.25 mg/mL Liberase TL for 90 min at 37 °C. After filtration through a 100 µm cell strainer, cell suspensions were stained with ZombieRed viability dye (423109, Biolegend, San Diego, CA, USA) per manufacturer’s instructions and then with conjugated antibodies on ice for 30 min. Stained cells were then washed and fixed with Cytoperm/Cytofix solution (BD Biosciences, San Jose, CA, USA) and acquired on an LSR Fortessa X-20 SORP. Data were analyzed using FlowJo software. anti-mouse CD16/32 (2.4G2) was from BioXCell (West Lebanon, NH, USA) (CUS-HB-197) and used at 1:300 dilution. anti-T-bet (4B10) (644803), anti-GATA3 (16E10A23) (653813), anti-mouse CD4 (GK1.5) (100449), anti-mouse CD3ε (145-2C11) (100327), anti-mouse CD45 (30-F11) (103105, 103111), anti-mouse NKp46 (29A1.4) (137617), anti-mouse/human CD11b (M1/70) (101245), anti-mouse CD11c (N418) (117328), anti-mouse Ly-6G (1A8) (127639), anti-mouse F4/80 (BM8) (123146), anti-mouse I-A^b (AF6-120.1) (116419) were from Biolegend and used at 1:300 dilution.

T. vaginalis was cultured overnight in complete Diamond’s media (26 (link)). The parasites were then pelleted by centrifugation and resuspended at 10⁶ cells/ml in the same media, with the exception that no horse serum was added (serum-free Diamond’s media), and incubated at 37°C at the time points indicated. At each time point, 500 µl of culture was taken from each sample and read on a FACS instrument. The survival rates of the parasites were determined by excluding dead cells using Zombie Red viability dye at a 1:1,000 dilution in PBS (BioLegend) and quantitated using CountBright Absolute Counting Beads (Thermo Fisher Scientific) with a BD LSRFortessa cell analyzer (see Fig. S2 in the supplemental material). The percentages of living cells at all of the time points were normalized to the level at time point 0 h, which was set as 100%.

HUVECs were grown to confluence in glass chamber slides and incubated with pooled patient plasma as described above. Cells were washed with 10 mM HBS (pH 7.4) containing 140 mM NaCl, 2.5 mM CaCl₂, and 2% FBS (annexin V binding buffer) and stained with annexin V-Alexa Fluor 488 (Thermo Fisher Scientific) at a 1:50 dilution and Zombie Red viability dye (BioLegend) at a 1:1000 dilution for 15 minutes at room temperature in annexin V binding buffer. Cells were washed and fixed in annexin V binding buffer containing 4% paraformaldehyde for 7 minutes. Cells were washed 3 times and mounted with DAPI. Images were obtained using a Zeiss LSM 880 upright laser scanning confocal microscope in 3 × 3 tile-scan mode with a Plan-Apochromat 20×/0.8 M27 objective.