Fortessa analyzer

Flow cytometry was performed as previously described (10 (link)). In brief, spleens and lymph nodes were pressed through a 70 μm strainer to generate a single-cell suspension, preincubated with 1G12 antibody to block nonspecific Fc binding, stained with directly conjugated antibodies for 15 minutes at 4°C, and washed twice. Antibodies and other reagents used for flow cytometry are listed in Supplemental Tables 2 and 3. Staining and washes were performed in PBS with 2% fetal bovine serum (FBS). FoxP3 staining was performed with an intracellular staining kit and according to manufacturer instructions (eBioscience, Thermo Fisher Scientific). Apoptosis was measured via annexin V–488 staining (Invitrogen, Thermo Fisher Scientific) for 15 minutes at room temperature in annexin staining buffer (BD Biosciences). APL formation was assessed using a CYTO-ID detection kit (Enzo Life Sciences). In some cases, transplanted animals were injected with BrdU 30 minutes prior to euthanization and samples processed according to manufacturer’s instructions (BioLegend 370706). Flow data were captured on a Fortessa analyzer (BD Biosciences) and evaluated using FlowJo software (version 10.1, Tree Star).

Synchronous cultures of our cMUT lines were treated with DMSO or RAP at ring stage, diluted to 0.1% parasitemia, and cultured in 96-well round-bottom plates for 3–4 cycles. Samples were collected at schizont stage on the cycle of treatment and at trophozoite stage on the following cycles, fixed for 30 min with 4% paraformaldehyde and 0.02% glutaraldehyde (Sigma) in PBSa at RT, diluted 5-fold in PBSa, and stored at 4°C. To determine the antiparasitic activity of HsSH inhibitors, a synchronous culture of P. falciparum 3D7 parasite at 1% parasitemia was treated at ring stage with a dose response of compounds and cultured for 72h in 96-well round-bottom plates. Samples were fixed as described above.
To determine parasitemia by flow cytometry, fixed samples were stained with 1 μg/mL of Hoechst 33342 (Thermo Fisher Scientific) in PBSa for 30 min at 37°C. Five to ten thousand RBCs per samples were analyzed by flow cytometry using a high-throughput Fortessa Analyzer (BD Biosciences) and the FACSDiva Software v8.0.1. Infected RBCs were distinguished from uRBCs as the population that showed positive DNA staining using a 355 nm UV excitation laser and a 450/50 nm emission filter. Flow cytometry data were analyzed using FlowJo LLC 2006–2015. For inhibitor treatment, EC₅₀ values were obtained by fitting the parasitemia to a dose response curve in Prism.