C6 accuri software

Splenocytes were prepared for flow cytometric analysis as previously described [32 (link)]. Splenocytes were stimulated with Tg ME49 Ag (4 μg/mL) for 2 hr at 37 °C with 5% CO₂. After antigen stimulation, cells were stained with fluorophore-conjugated GL7 (PE) and B220 (FITC) antibodies purchased from BD Biosciences (Franklin Lakes, NJ, USA) and Invitrogen (Waltham, MA, USA). All flow cytometry procedures were performed according to the manufacturer’s protocol. Stained samples were analyzed using the Accuri C6 flow cytometer and the C6 Accuri software (BD Biosciences, Franklin Lakes, NJ, USA).

To evaluate cell death, 50,000 cells were seeded in a 24-well plate for 24 h at 37 °C and 5% CO₂ for complete adherence and treated with EcTI (5–100 µM) in the experimental groups or fresh medium in the control group. For cytometric analysis, cells were detached using Versene solution (0.2 g EDTA/liter of PBS; Thermo Fisher Scientific Inc., Waltham, MA, USA) and washed with PBS. The suspension was labeled with cell death staining solution (2.5 µg/mL annexin-FITC and 5 µg/mL PI in binding buffer (0.1 M HEPES/NaOH, pH 7.4, 1.4 M NaCl plus 25 mM CaCl₂)) according to the manufacturer’s instructions (Annexin V/Dead Cell Apoptosis Kit, Thermo Fisher Scientific). The cells were incubated for 15 min at room temperature, protected from light, and at least 10,000 events were collected for acquisition. Dot plot analyses were performed in a BD Accuri C6 Cytometer using C6 Accuri Software (BD, Los Angeles, CA, USA). Staurosporine (1 µM) was used as the apoptosis control, and Triton X-100 (0.1%) was used as the necrosis control [40 (link)].

Flow cytometry was performed on splenocytes and MLN cells as previously described [17 (link)]. Splenocytes (1 × 10⁶ cells/mouse) and MLN cells (1 × 10⁵ cells/mouse) were stimulated with T. gondii ME49 antigen (2 μg/mL) at 37 °C with 5% CO₂ for 2 h. Stimulated cells were stained with fluorophore-conjugated antibodies purchased from BD Biosciences (Franklin Lakes, NJ, USA) and Invitrogen (Waltham, MA, USA). CD4⁺ T cells, CD8⁺ T cells, and germinal center B (GC B) cell populations were measured using the following antibodies: CD3 (PE- Cy7), CD4 (FITC), CD8 (PE), GL7 (PE), and B220 (FITC). All staining procedures were performed according to the manufacturer’s protocol. Stained cells were acquired by Accuri C6 flow cytometer and analyzed with the C6 Accuri software (BD Biosciences, Franklin Lakes, NJ, USA). For GC B cells, B220⁺ lung cells and splenocyte populations were gated, and from this population those positive for GL7 were gated. For CD4⁺ and CD8⁺ T cells, CD3-positive single cell populations were gated and from this population CD4⁺ and CD8⁺ cells were determined.

Splenocytes and MLN cells were prepared for flow cytometric analysis as previously described (Kang et al., 2020a (link)). Single cell suspensions of splenocytes (1 x 10⁶ cells/mouse) and MLN cells (1 x 10⁵ cells/mouse) were stimulated ex vivo with T. gondii ME49 antigen (2 μg/mL) at 37°C with 5% CO₂ for 2 hours. After antigen stimulation, cells were stained with the following fluorescent-conjugated antibodies purchased from BD Biosciences (Franklin Lakes, NJ, USA) and Invitrogen (Waltham, MA, USA) for CD4⁺ T cell, CD8⁺ T cell, and germinal center B (GC B) cell detection: CD3 (PE-Cy7), CD4 (FITC), CD8 (PE), GL7 (PE), and B220 (FITC). All staining procedures for flow cytometry were performed according to the manufacturer’s protocol. Stained cells were acquired using the Accuri C6 flow cytometer and analyzed with the C6 Accuri software (BD Biosciences, Franklin Lakes, NJ, USA).

After siRNA transfection or drug treatments for 72 h, 10⁵ cells were collected, washed in phosphate-buffered saline (PBS), pelleted by centrifugation and fixed in 70% ethanol. Immediately prior to staining, cells were suspended in PBS containing 50 μg/ml of RNAse A (Qiagen, UK), and then, stained with propidium iodide (final concentration 100 μg/ml) overnight at 4°C. The percentage of cells in subG₁, G₀/G₁, S and G₂/M phases were determined from 10.000 cells using the BD C6 ACCURI Software (Becton Dickinson). The experiments were carried out three independent times (triplicates).