Cytexpert software version 2

Induction of apoptosis was examined using Annexin-V FITC/PI stains and a similar experimental procedure for the cell cycle was followed apart from the apoptosis analysis, which did not require fixation of cells with 70% ethanol. Instead of ethanol fixation, the cell number of each sample was counted, and the cells were diluted to 1 × 10⁵ cells with 1 mL PBS in fresh FACS tubes. Cells were centrifuged and then resuspended in 1 mL of cold PBS (the whole step was repeated twice). Pelleted cells were resuspended in 100 μL binding buffer, and subsequently, 3 μL of Annexin V and 3 μL of PI were added to all tubes and incubated in the dark for 15 min at room temperature. Following the incubation period, 200 μL of 1 x binding buffer was added. Samples were assessed using a Cytoflex flow cytometer (Beckman Coulter, Brea, CA, USA) within 1 h of staining and data were analysed using CytExpert software version 2.1 (Beckman Coulter, Brea, CA, USA).

For the determination of the mitochondrial membrane potential in the promastigote forms of L. amazonensis, we used tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes, Carlsbad, CA, USA) and flow cytometry. For this method, 2 × 10⁶ parasites/mL were treated with (-)-5-demethoxygrandisin B IC₅₀ for 24 h at 26 °C. Nontreated parasites were used as a negative control, and heat-killed parasites (60 °C bath for 30 min) were used as a positive control. Post-incubation, the parasites were subjected to centrifugation at 1500× g for 5 min at room temperature. Afterward, they were washed with PBS and incubated in a solution of 300 μL TMRE (50 nM) in the absence of light for 15 min at room temperature. Subsequently, a flow cytometry analysis was conducted using a CytoFlex flow cytometer (Beckman Coulter Life Sciences, Inc., Brea, CA, USA). TMRE excitation was achieved using a 488 nm blue laser, and the emitted fluorescence was captured with a 585/42 nm bandpass filter. The flow cytometry data were analyzed using CytExpert software version 2.1 (Beckman Coulter Life Sciences, Inc., Brea, CA, USA) [26 (link)].

The promastigote forms of L. amazonensis (2 × 10⁶ cells/ml) were treated with monomethylsulochrin IC₅₀ for 24 h at 26°C. Heat-killed parasites (60°C bath for 60 min) and untreated parasites were used as positive and negative controls, respectively. After incubation, parasites were centrifuged at 210g for 5 min at room temperature, washed in PBS, incubated with 300 μl of TMRE (50 nM) in the dark for 15 min, at room temperature, and submitted to flow cytometry through CytoFlex flow cytometer (Beckman Coulter Life Sciences, IN, USA). TMRE was excited by 488-nm blue laser, and its fluorescence was collected at 585/42 nm bandpass filter. CytExpert software version 2.1 (Beckman Coulter Life Sciences, IN, USA) was used for flow cytometry analyses (Mondêgo-Oliveira et al., 2021 (link)).

The effect of rilpivirine on cell cycle distribution in T47D breast cancer cells was evaluated by flow cytometric analysis. Briefly, cells were seeded in 6-well plates at a density of 6 × 10⁴ cells/well and then plates were incubated overnight at 37 °C in a 5% CO₂ incubator. Following the incubation, the test compound was added to individual wells and the plates were incubated for 48 and 72 h. Subsequently, the medium was removed from the wells and transferred into fluorescence-activated cell sorting (FACS) tubes. The adherent cells remaining were trypsinised, re-suspended in media, and transferred into the FACS tubes. Cells were centrifuged, fixed with cold 70% ethanol for 15 min, and pelleted. The pelleted cells were then re-suspended in 200 μL propidium iodide (PI) solution (50 μg/mL propidium iodide, 0.1 mg/mL ribonuclease A, 0.1% sodium citrate, 0.1% Triton X-100) and incubated in the dark for 1.5 h at room temperature. Following the incubation, 200 μL PBS was added. Samples were assessed using a flow cytometer (Cytoflex; Beckman Coulter Inc, Brea, CA, USA), and data were analysed using the CytExpert software version 2.1 (Beckman Coulter, Brea, CA, USA).

Stemness of PN-transfected BCA cells was determined by staining with anti-CD24 and anti-CD44 antibodies and analyzed by flow cytometry and compared with mock-transfected cells in conditions without or with anti-PN peptide. Briefly, BCA cells were seeded at the concentration 1 × 10⁵ cells into 6-well plates with 2 ml of complete media. Cell media was removed the next day and changed to 1% FBS media for 24 h. Then, the treatment in 1% FBS media without or with anti-PN peptide (1 μM) were refilled and incubated for a further 24 h. At the end of treatment, cell pellets were collected and incubated in 2% FBS/1x PBS with 1:20 dilution of FITC-labeled anti-CD24 antibody (cat no. 21270043, ImmunoTools GmbH, Friesoythe, Germany) and 1:5 dilution of allophycocyanin (APC)-labeled anti-CD44 antibody (cat no. 21270446, ImmunoTools) for 30 min. CytoFLEX® Flow cytometry (Beckman Coulter, Inc. Brea, CA, USA) and CytExpert® software version 2.1 (Beckman Coulter, Inc.) were used for analysis.

To measure the mitochondrial membrane potential, promastigote forms of L. amazonensis (2 × 10⁶ parasites mL⁻¹) were treated with carajurin for 24 h with calculated IC₅₀, in Schneider’s Insect Medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin. Heat-killed parasites (60 °C bath for 30 min) were used as positive control and nontreated parasites were used as a negative control. Subsequently, the parasites were incubated for 30 min at 26 °C with 50 nM tetramethylrhodamine, followed by ethyl ester (TMRE) for 15 min at room temperature, and submitted to flow cytometric analysis through a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Inc., Brea, CA, USA). TMRE fluorescence was excited through a 488 nm-blue laser and their fluorescence was collected at 585/42 bandpass filter. CytExpert software version 2.1 (Beckman Coulter Life Sciences, Inc., Brea, CA, USA) was used for flow cytometric analyses.