Cyan adp flow cytometer

Cells were fixed with Cytofix/Cytoperm Fixation solution (BD Biosciences) for 20 min at 4°C, then washed with Perm Wash Buffer/phosphate-buffered saline (PBS, 1 × ; BD Biosciences) and permeabilized with Perm Wash Buffer/PBS +0.1% Triton X-100 for 30 min. Cells were blocked with 3% BSA in 1 × Perm Wash Buffer at room temp for 30 min. After blocking, cells were incubated in primary antibody (Supplementary Table S2) diluted in 1 × Perm Wash Buffer +0.1% Triton X-100 4°C for 45 min. Alexa Fluor^®-tagged secondary antibody was added after primary incubation for 1 h at room temperature. Samples were run on a Beckman Coulter CyAn-ADP flow cytometer, and subsequent datasets were analyzed using FlowJo software.

Promastigote form of the endogenous tagged cell lines, in logarithmic phase, at 5 × 10⁵ cell mL⁻¹ were treated with different miltefosine concentrations during multiple time courses at 25 °C. After the treatment, cells were washed once at 2000 g for 5 min with PBS at room temperature and resuspended in 500 μL of PBS. Cells were analysed for FACS using a Beckman Coulter CyAn ADP flow cytometer and Median Fluorescence Intensity (MFI) were measured by FlowJo™ 10.6.2 software. The untagged parenteral T7/Cas9 cell line (P) was used as control to subtract auto fluorescence and protein fold change level was calculated as follow: MFI_{(fold change)} = [(MFI_{tagged_treated}) - (MFI_{P_treated})]/[(MFI_{tagged_untreated}) - (MFI_{P_untreated})].

Cell cycle profile analysis was performed using the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. Briefly, cultured cells were incubated at 37 °C with 10 μM EdU for 1 h and harvested after dissociation with TrypLE. After 3 washes with 0.1% BSA-PBS, cells were fixed with 1% paraformaldehyde for 15 min at room temperature and washed three more times with 0.1% BSA-PBS. Cells were then permeabilised for 15 min with saponin-based permeabilisation/wash buffer and incubated with the Click-iT reaction cocktail for 30 min protected from light. Cells were washed once with saponin-based permeabilisation/wash buffer, stained for DNA content using DAPI (ThermoFisher Scientific), and analysed on the Cyan ADP flow cytometer and FlowJo VX software.

To prepare single cell suspension, muscles were dissected from mice and finely minced with a scalpel in a dish containing DMEM, as previously described [12 (link)]. Next, minced muscles were digested in a solution of Collagenase type IV (Worthington, Lakewood, NJ, USA) 1 mg/mLin DMEM, (10 mL/g of muscle) for 1h 30 min in shaking water bath at 37 °C. Then, digested muscles were passed through a 70 µm cell strainer first and then through a 40 µm cell strainer, to exclude cell debris. Muscle single cell suspension was then resuspended in FACS buffer (PBS 1% FBS) and incubated 30 min on ice with the following antibodies: anti-CD45 (Biolegend, clone 30-F11, 1:6000), anti-Ly6G (eBioscience, clone 1A8 and clone RB6-8C5, 1:3000), anti-Ly6c (Biolegend, clone HK1.4, 1:100), anti-F4/80 (Biolegend, clone BM8, 1:1000), anti-CD206 (MMR) (Biolegend, clone C068C2, 1:50), anti-CD11b (Biolegend, clone M1/70, 1:3000). Cell viability was assessed with 4′,6-diamidino-2-phenylindole dilactate (DAPI) (BioLegend).
For CFSE analysis isolated SCs were stained with CFSE (ThermoFisher Scientific, Waltham, MA, USA) 5 µm for 20′ at 37 °C in dark prior to culture. Samples were processed using a Dako CyAn ADP flow cytometer and acquired data were analyzed using FlowJo software version 10 (FlowJo LLC, Ashland, OR, USA).

Cell cycle profile analysis was performed using the Click-iT EdU Pacific Blue Flow Cytometry Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. In summary, cultured cells were incubated at 37°C with 10 μM EdU (5-ethynyl-2′-deoxyuridine) for 1 hr and harvested using cell dissociation buffer (Gibco). After three washes with PBS/1% BSA, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed three more times with PBS/1% BSA. Cells were then permeabilized for 15 min with saponin-based permeabilization/wash buffer and incubated with the Click-iT reaction cocktail for 30 min protected from light. Cells were washed once with saponin-based permeabilization/wash buffer and stained for DNA content using the FxCycle Far Red dye (Invitrogen). Cells were analyzed on the Cyan ADP flow cytometer and FlowJo software.