Facsverse

Accurate cell counts of lymphocyte cultures were taken by using AccuCheck counting beads (Life Technologies, UK) or, alternatively, by direct event counts against volumetric flow rate on FACSVerse (Beckton Dickinson). Antibodies used for flow cytometry were conjugated to fluorescein-isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll protein (PerCP)-Cy.5.5, PE-Cy7, allophycocyanin (APC), APC-eFluor 780 or Alexa Fluor 647 and were obtained from BD Pharmingen or eBiosciences unless otherwise stated: anti-CD8α (clone 53-6.7), anti-CD25 (clone PC61), anti-CD44 (clone IM7), anti-CD69 (clone H1.2F3), CD71 (clone C2), CD98 (clone RL388; Biolegend). Intracellular levels of S6 protein phosphorylated on S235 and S236 (S6^S235/6) were detected by using Alexa-Fluor-647-conjugated anti-S6^S235/6 (4851; Cell Signalling Technologies) as previously described [23] . Following incubation with antibodies, cells were washed and re-suspended in FACS buffer. Samples were analysed using LSR II Fortessa or FACSVerse (Becton Dickinson). A minimum of 1×10⁴ ungated events were acquired and stored. Data files were processed using the FlowJo software V9.6.4 (Treestar) for Mac OS. Live cells were gated according to their forward and side scatters.

Cells were incubated with different concentrations of DCZ0415 for 24 h.
For cell cycle assay: cells were harvested, followed by washing twice with PBS, and were fixed overnight with 70% ethanol. Cell samples were stained by propidium iodide (PI), and the intensities were detected using flow cytometry (FACSVerse, BD, USA) by Analysis and Testing Center, Nanjing Medical University.
For apoptosis assay: supernatant DMEM medium and cells were collected, centrifuged 3 min, 800 g, and the pellets were washed twice by PBS. Cells were resuspended in 400 μL of precooled 1×binding buffer and stained by Annexin V-FITC/PI (Yeasen, Shanghai, China) for 30 min. Then cell suspension was detected on flow cytometry (FACSVerse, BD, USA) analysis and Testing Center, Nanjing Medical University.

Total mitochondrial mass and mitochondrial transmembrane potential (Δψ_m) were analyzed by incubating cells with 50 nM MitoTracker Green FM or 200 nM TMRM (Thermo-Fisher Scientific), respectively, in DMEM without serum and phenol red (Thermo-Fisher Scientific) for 30 min. Stained cells were washed twice with cold PBS, collected and analyzed by flow cytometry (FACS Verse, BD Biosciences). Normalized Δψ_m was calculated as TMRM/MitoTracker Green FM relative fluorescence (geometric mean). Reactive oxygen species (ROS) were analyzed by incubating cells for 15 min in serum-free DMEM with 5 μM of the superoxide indicator dihydroethidium (DHE, Thermo-Fisher Scientific), or, alternatively, with 5 μM of 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA, Thermo-Fisher Scientific), a chemically reduced form of fluorescein used as a general indicator for ROS. Stained cells were washed twice with cold PBS, collected and analyzed by flow cytometry (FACS Verse, BD-Biosciences). For both probes, fluorescence intensity (geometric mean) was recorded (DHE, Ex/Em: 510/580 nm; H₂DCFDA Ex/Em: 495/517 nm) and values were expressed as relative fold change with respect to normal cells.

Division index was measured using carboxyfluorescein succinimidyl ester (CFSE) staining. Lin-CD34+ or MEG-01 cells were labeled by CFSE according to the manufacturer’s instructions and culture with or without EC as described above. At 24, 48 and 72 h, adherent and non-adherent co-cultured cells and control hematopoietic cells were analyzed using flow cytometry (FACSVerse; BD). The division index was quantified according to CFSE signal intensity using FlowJo software.
The CD34+CD38+ and CD34+CD38- populations cells were analyzed throughout time of culture using antiCD31-PB, antiCD34-PECy7, antiCD38-APC and CFSE multi-staining. The cell cycle assay was performed as described previously [30 (link)]. Briefly, cells were fixed with cold paraformaldehyde at 4% for 20 min at 4 °C. They were washed twice with staining buffer (PBS with 3% of FBS) and the cell membrane staining performed. The cells were incubated with antiCD34-PECy7, antiCD38-APC and antiCD31-PB for 30 min at room temperature, and were incubated with permeabilization solution (0.1% Triton X-100, 10% FBS in 1X PBS) for 15 min at room temperature. Cells were washed and incubated with antiKi67-FITC for 30 min and with DAPI (2 $\mathsf{\mu }$ M) for 10 min at room temperature to finally be analyzed by multiparametric flow cytometry (FACSVerse; BD).

Three groups of cells were treated with or without DFO for 24 h. After treatment the cells were trypsinized and washed twice with PBS and resuspended with 500 μl binding buffer. Then 5 μl of Annexin V-KeyFlour647 antibody and 5 μl of 7-ADD was added, and incubated for 15 min at room temperature in the dark according to the manufacturer’s protocol prior to FACS analysis. Both early and late stages of apoptotic cells were detected by a flow cytometer of FACSVerse™ (BD, USA).
Mock, Con and RNAi cells were plated in 6-well plates (1×10⁵/well) after reaching 80% confluence; the cells were treated with or without DFO, and then incubated for 24 h at 37°C in a humidified atmosphere with 5% CO₂. Cells were washed in ice-cold PBS and harvested by trypsinization. The cells were washed with ice-cold PBS twice and fixed with 75% pre-cold ethanol overnight at 4°C. The cells were centrifuged at 2,000 rpm for 5 min, the supernatant was discarded. One hundred microliters of RNaseA (200 μg/ml) was added to the cells for 30 min at 37°C, then 400 μl propidium iodine (10 μg/ml) was added to the cells for 30 min at 4°C in the dark. Flow cytometry was used for analysis by FACSVerse™ (BD).

Bone marrow cells were harvested 1 h after intraperitoneal injection of BrdU (100 mg/kg), and subsequent incubation with APC-anti-CD11b (M1/70), FITC-anti-CX3CR1 (SA011F11), BV421-anti-cfms (AFS98) antibodies (BioLegend), and PE-Cy7-anti-Ly-6C (AL-21), BV510-anti-BrdU (3D4) antibodies (BD Biosciences). Immunostained cells were prepared for analysis of BrdU incorporation using the FITC BrdU Flow Kit (BD Biosciences) by flow cytometric analysis using the FACS Verse (BD Biosciences). Data were analyzed by FACS Suite software (BD Biosciences). For analysis of the cell cycle of primary osteoclasts in vitro, cells were cultured with 10 μM BrdU for 45 min, and prepared for analysis of BrdU incorporation using the FITC BrdU Flow Kit. Cell death assay was performed with FITC-Annexin V (BD Biosciences) and Propidium Iodide (PI) by FACS Verse.

Cell cycle was assessed using propidium Iodide staining and FACS as described [54 (link)]. HCC827 cells were seeded in quadruplicates in 6-well plates and incubated overnight. Cells were treated with 0, 10, or 20 mM of 2DG in medium supplemented with 1 μg/mL puromycin, 1.5 mM pyruvate, and 0.05 mg/mL uridine. Samples were collected after 24 hours and fixed in 70% ethanol. Fixed cells were treated with RNase and PI for 2 hours and analyzed by flow cytometry (BD Biosciences FACS Verse). The FITC Annexin V apoptosis detection kit (catalog number 556547; Becton Dickinson) was used per manufacturer’s instructions, and cells were analyzed by flow cytometry (BD Biosciences FACS Verse). The results were analyzed using FlowJo V.10.1 software (FlowJo, LLC, Ashland, OR).