Facs software

HeLa cells, seeded in 6-well plates at a cell density of 6 × 10⁴ cells/well in 1.5 mL of culture medium, were incubated for 24 h, then the medium was replaced with a C₆₀-containing medium. After 24 h of incubation with C_60, HeLa cells were treated with US, as indicated above. At 24 h after US treatment, cells were harvested. Apoptosis was detected by Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit according to the manufacturer’s instructions. Briefly, cells were harvested and washed with binding buffer. After the addition of FITC-conjugated Annexin V, cells were incubated for 15 min at room temperature in the dark. Cells were washed with Binding buffer and, at 10 min after propidium iodide addition, were analyzed with the BD FACSJazz™ (BD Biosciences, Singapore). A minimum of 2 × 10⁴ cells per sample were acquired and analyzed with the BD FACS™ Software (BD Biosciences, Singapore).
On every histogram of flow cytometry four populations of cells are presented according to green (Annexin V-FITC) and red propidium iodide (PI) fluorescence intensities: viable (Annexin V-FITC negative, PI negative), early apoptotic (Annexin V-FITC positive, PI negative), late apoptotic (Annexin V-FITC positive, PI positive) and necrotic (Annexin V-FITC negative, PI positive) cells.

Single cells were index sorted into 96-well plates without any specific staining selection using the FACS AriaII (BD Biosciences). The plates were pre-filled with 20 µl reverse crosslinking buffer (see above) per well. For each experiment, index sort files (fcs) were exported using BD FACS Software and then further analyzed by FlowJo and R.
For reverse crosslinking, all 96-well plates were incubated overnight at 65 °C, then proteinase K was inactivated by 10-min incubation at 80 °C.
Single-cell libraries were prepared by adding 25 µl 2 × PCR Master Mix (NEBNext High fidelity, NEB) and 2.5 µl of 25 mM barcoding primer to each well. PCR cycling conditions: 72 °C for 5 min; 98 °C for 30 s; 20 cycles at 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min. After that, all wells were pooled, purified using the MinElute kit from Qiagen and eluted in 20 µl Qiagen EB elution buffer. The 75 × 2 paired-end sequencing was performed on Illumina HiSeq4000 at the Stanford Functional Genomics Facility. On average, we sequenced ~1000 single cells on one HiSeq 4000 lane, resulting in an average of ~0.3 millions reads per cell.

0.5-1 million cells of each single-cell suspension was incubated with the respective conjugated antibody for 15 min at 4°C and analyzed with a BD LSR. Unstained cells were included as negative controls for each FACS analysis. The antibodies used in this study were: anti-CD133-PE (eBioscience). The data were analyzed using BD FACS software.

At 24 h following transfection or inhibitor treatment, the indicated cells were treated with cisplatin at a final concentration of 10 μM. After 24 h of cisplatin treatment, the floating and attached cells were collected separately and then combined. 0.5–1 million cells of each single-cell suspension was stained with the Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), and analyzed with a BD LSR. Unstained cells were included as negative controls for each FACS analysis. The data were analyzed using BD FACS software.

EPCs are cells positive for endothelial markers, such as VEGF receptor 2 (VEGFR2), and hematopoietic stem cell markers, such as CD34 [5 (link), 15 (link)]. We used immunofluorescent staining for CD34 and the VEGFR2 to identify mononuclear cells (MMNs) positively in blood as CEPCs. We incubated whole fresh blood samples anticoagulated with EDTA.2Na with PE-conjugated anti-CD34 antibody and FITC-conjugated anti-VEGFR2 antibody to identify the CD34⁺/VEGFR2⁺ cells among the MNCs. We dissolved the red blood cell lysate in distilled water and applied it to the cells at a final concentration of 10% (v/v).
To count the EPCs per milliliter of blood, two-color cytometry analysis of the samples was performed on a cytometer equipped with a four-color option (BD FACS Jazz). EPCs were detected by gate analysis excluding events with different origins, such as non-specific staining events and non-hematopoietic circulating cells. The data were collected from 100,000 cells for each sample and analyzed with BD FACS software.

H9C2 cells (2 × 10⁵) were plated in 6‐well plates for 24 hours. After different treatments (note: LY294002 and SDX added at the same time), cells of each well were collected, washed twice with PBS and resuspended with 200 µL PBS. Then cells (100 µL) were incubated with 5 µL annexin V‐fluorescein isothiocyanate for 5 minutes, followed by 3 µL propidium iodide (PI) for 10 minutes in the dark at room temperature. The fluorescence intensities of annexin V/PI‐stained cells were analysed by a flow cytometry. The apoptosis ratio was quantified by BD FACS software.