Fc500 mpl flow cytometer

Cells were harvested by trypsinization (0.05% (w/v); Life Technologies, Cat. #15400-054) for 2 min, and the cell pellet was resuspended in PBS to a titer of 10⁶/100 μL. The cell suspension was incubated with the following antibodies respectively. CD34-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany, Cat. #130-098-142), CD45-PE (Miltenyi Biotec, Cat. #130-080-201), CD73-PE (Miltenyi Biotec, Cat. #130-112-060), CD90-FITC (Miltenyi Biotec, Cat. #130-095-403) and CD105-FITC (Miltenyi Biotec, Cat. #130-112-327). About 2 μL of each antibody (1:10 diluted according to the datasheet of the antibodies) per tube was added and incubated for 10 min in darkness at 2–8 °C. Cells were washed with PBS completely and resuspended in 1% (w/v) paraformaldehyde. Samples were run on a FC500MPL flow cytometer (Beckman Coulter, Brea, CA, USA) and the data were analyzed by FlowJo vX.0.7 software (FlowJo LLC, Ashland, OR, USA).

At sacrifice, cell suspensions were obtained from the harvested immune organs and tumours using mechanical disruption in PBS 1X-BSA 0.5%. They were filtered using a 40 μm-pore filter (Falcon^® 40 µm Cell Strainer) and submitted to hypertonic RBC lysis. Cells were counted by flow cytometry using Flow-Count Fluorospheres (Beckman Coulter, Hialeah, FL, USA). Cell suspensions were diluted with staining buffer (PBS 1X—BSA 0.5%—EDTA 2 mM) and 1 × 10⁶ cells were deposited into wells of a U-bottom-shaped 96-well plate. Cells were surface-labelled with specific antibodies for 30 min at 4 °C, as indicated in Table S2. Intracellular staining using anti-FoxP3-Biotin was performed according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA). Dead cell exclusion was performed using 10 µg/mL propidium iodide labelling. Fluorescence was quantified with a four-colour Beckman-Coulter FC500 MPL Flow Cytometer (Beckman-Coulter, Hialeah, FL, USA). Data files were analysed with Kaluza 1.2 software (Beckman-Coulter, Hialeah, FL, USA). The FACS gating strategy is illustrated in Figure S1: compensations and controls used the FMO (Fluorescence Minus One) procedure with corresponding antibody isotypes. Due to the constraints of the methods, half of the animals were randomized and used in cytometry.

Cells were seeded at a density of 2 × 10⁵ (MDA-MB-231) and 3 × 10⁵ (HUVECs) cells/well in 6-well plates, respectively, and allowed to attach for 24 h. MDA-MB-231 were treated with 10 µg/mL of native sEVs, 20 µg/mL of BRB, or 5 µg/mL of sEV-loaded BRB, whereas HUVECs were treated with 10 µg/mL of native sEVs, 40 µg/mL of BRB, or 25 µg/mL of sEV-loaded BRB. After 24 h, cells were washed in PBS, before adding 0.2 mL of nuclear isolation medium 0.6% NP40, 50 µg/mL propidium iodide, 100 µg/mL RNase in PBS (all from Sigma Aldrich). Cells were then incubated in the dark for 60 min and analyzed with FC 500 MPL flow cytometer (Beckman Coulter).

Logarithmically growing promastigotes (1×10⁶ cells) were incubated with Mitotracker Red CMXRos (Life Technologies) to a final concentration of 100 nM and maintained under normal growing conditions for 45 minutes. As a mitochondrial membrane depolarization control the protonophore carbonyl cyanide m-chloro phenyl hydrazone (CCCP) was used to a final concentration of 100 µM under the same conditions. Subsequently, samples were analyzed using a FC500 MPL flow cytometer (Beckman-Coulter).

Cells were removed from the dish with Versene and washed twice with fresh medium before counting using a haemocytometer. Cells were resuspended at 1 × 10⁵ cells/mL in Binding buffer (0.5 M HEPES, pH 7.4; 2% fetal calf serum; 2 μg/mL IgG) and 8 μg of primary antibody added before incubation on ice for 1 h. Cells were washed twice in cold Binding buffer before the addition of 0.5 μg of goat anti-mouse-FITC secondary antibody and incubation on ice for 1 h. Cells were again washed twice in ice-cold Binding buffer and the pellet resuspended in 600 μL of FACS Fix solution (1% paraformaldehyde and 1% glucose in PBS). Analysis of the cells was performed using a Beckman Coulter FC500 MPL Flow cytometer.

Pancreatic cancer cells were incubated for 72 h in 60 mm
² culture dishes with glutamine-free medium, medium containing 2 mM glutamine, or medium containing 2 mM glutamine supplemented with 50 μM diazooxonorleucine (Cat. #HY-108357; MedChemExpress, Shanghai, China) or 10 μM azaserine (Aza; Cat. #HY-B0919; MedChemExpress). Then, the cells were incubated with 10 μM 2’,7’-DCFH-DA (Cat. #D6883; Sigma-Aldrich, St Louis, USA) in serum-free DMEM at 37°C for 30 min. Cells treated with 100 μM H
₂O
₂ were used as positive controls. Thereafter, cells were treated with glutamine-free medium containing 2 μM ferrostatin-1 (Cat. #HY-100579; MedChemExpress), 5 mM N-acetylcysteine (NAC, Cat. #HY-B0215; MedChemExpress), 50 μM Z-VAD-FMK (Cat. #HY-16658B; MedChemExpress), and 150 μM necrostatin-1 (Cat. #HY-15760; MedChemExpress) to counteract cell death. The cells were then washed twice, resuspended, and filtered into single-cell suspensions before analysis. The fluorescence intensity of DCFH, formed by the reaction of DCFH-DA dyes with ROS, was measured at excitation and emission wavelengths of 488 and 530 nm, respectively, with a FC500 MPL flow cytometer (Beckman Coulter, Pasadena, USA). A minimum of 10,000 cells were analyzed per condition
[13] (link). Proportion analysis and histogram generation were performed using FlowJo software (version 10.6; FlowJo, Ashland, USA).