Accuri c5 cytometer

FITC-labeled anti-CD80, PE-labeled anti-CD86 antibodies were obtained from BD Pharmingen (San Diego, CA, USA). Samples were analyzed using BD Accuri™ C5 Cytometer (Becton, Dickinson and Company, San Jose, CA, USA) and the data were analyzed using FlowJo version 10.0 (Tree Star Inc., Ashland, OR, USA). Inflammatory cytokines (IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12p70 protein levels in a single sample) were determined in cell-free supernatants with the BD™ CBA Human Inflammatory Cytokines Kit (Becton, Dickinson and Company).

Epismatigotes forms of T. cruzi (Dm28c strain) were grown at 28°C in BHI medium at a concentration of 2x10⁶ cells.mL^-1 in 12-well culture plates (TPP^™, Switzerland). Test compounds (CUR, DMC and BMDC) were added in triplicate at concentrations of 10, 50 and 100 μM. The controls used were the vehicle (DMSO 0.02%) and paclitaxel at 20 μM concentration. After 48 h of incubation, parasites were fixed in ethanol 70% for 30 min at -20°C. Next, cells were centrifuged at 200 rcf for 8 min and washed with PBS. Then, the samples were incubated for 10 min with a permeabilization buffer (0.2 M Na₂HPO₄ and 0.005% Triton-X, pH 7.8). Finally, cells were centrifuged at 200 rcf for 8 min and stained in a solution of 50 μg.mL^-1 propidium iodide (PI) and 0.2 μg.mL^-1 RNAse for 30 min at room temperature. DNA content was analyzed measuring PI fluorescence in a BD Accuri C5 cytometer (Becton Dickinson Company, USA) at 488 nm excitation wavelength and 585/40 nm emission filter.

After transfection, SL were incubated for 67 h at 37°C in the 5% CO₂, and parasite load was determined by flow cytometry. The method described by [26 (link)] was used, with modifications. SL (10⁴ cells / treatment) were fixed with 1% paraformaldehyde for 60 min at room temperature and permeabilized in ethanol for 60 min at -20°C. SL were incubated with Leishmania gp63 monoclonal antibody mouse IgG2a non-conjugated (ABD, Serotec, USA) at 4°C for 60 min. After three successive washes with PBS at pH 7.2 with 2% BSA (bovine serum albumin), cells were stained with secondary antibody goat anti-mouse IgG2a conjugated to phycoerythrin (PE) (R&D Systems, USA), and monoclonal antibody anti-human CD14 conjugated to fluorescein isothiocyanate (FITC) (Bio Rad, USA) for 60 minutes at 4°C. After incubation with secondary antibody, SLs were washed in PBS at pH 7.2 with 2% BSA, and resuspended in PBS at pH 7.2. Flow cytometry was performed on an Accuri C5 cytometer (BD Biosciences, USA). Acquisition of 10,000 events was counted for each replicate on channel FL1 and FL2, and data analysis was performed using the BD Accuri C6 software (version 1.0.264; BD Bioscience, CA, USA). Cells were gated on monocytes (CD14+ cells) and positivity for gp63+ was considered in the analysis.