Annexin 5 fitc apoptosis detection kit

The number of apoptotic cells was measured by flow cytometry using the Annexin V-FITC apoptosis detection kit (Roche, Germany). Approximately 1–5 x 10⁵ cells were acquired in each sample. Samples were analyzed by bivariate flow cytometry using a BD FACSCanto cytometer equipped with FlowJo software.
TUNEL staining was performed using the In Situ Cell Death Detection Kit-TMR red (Roche, Germany) following the manufacturer’s instructions. Both TMR (red) and DAPI (blue) fluorescence were visualized and imaged using an IX53 inverted phase contrast microscope (OLYMPUS Inc., Japan).

Cell apoptosis assay was conducted by flow cytometry with the Annexin V-FITC Apoptosis Detection Kit (Roche, China). Briefly, cells were trypsinized, neutralized and washed with PBS. Cells were resuspended in binding buffer and incubated with Annexin V-FITC and propidium iodide for 10 min. Then cells analyzed by BD FACSCalibur (BD Bioscience, USA) and analyzed by Flowjo software (FlowJo, USA). The experiment was repeated three times.

Apoptotic cells population was determined using PI-Annexin V assay (Annexin V-FITC Apoptosis Detection Kit Roche). 5×10⁴ cells/well were seeded in 24-well plate. Cells were treated with various concentrations of samples either single or in combination for 24 h. Cells were harvested, added with 1× binding buffer, labeled with PI-Annexin V, and incubated at room temperature in the dark for 5 min. The cells suspension was analyzed using a flow cytometry (Becton Dickinson) and followed by flowing software (version 2.5.1) and Excel MS Office 2013.

The effect of miR-381 on cell apoptosis was assessed using Annexin V-FITC apoptosis detection kit (Roche Applied Science, Germany) following the manufacturer's instructions. Briefly, MCF-7 and MDA-MB-231 cells at a density of 3×10⁵ were seeded into a 6-well plate and incubated for 24 h at 37 °C, with 5 % CO₂. Subsequently, cells were transfected with miR-381 mimic, miR-381 inhibitor and their NCs as described above. After 48 h, the media was aspirated and cells were treated with accutase enzyme (200 µL/well) for 5 min to dissociate the adhered cells. The protease activity was stopped by adding some medium containing FBS and the cells were collected by centrifugation. After removal of supernatant, cells were washed twice with PBS and incubated with Annexin V-FITC and propidium iodide (PI) for 15 min. The stained cells were detected and quantified using FacsCalibur (Becton Dickinson, San Jose, USA) flow cytometer equipped with 488 nm laser for excitation, and 515 nm and 600 nm band-pass filters for the detection of FITC and PI, respectively. The obtained data were analyzed using CellQuest software (BD Biosciences, US). Annexin V-FITC positive/ PI negative cells were reported as early apoptotic cells.

Induction of apoptosis was
determined by a flow cytometric assay with Annexin V–fluorescein
isothiocyanate (FITC) by using an Annexin V–FITC apoptosis
detection kit (Roche). Exponentially growing HeLa cells in 6-well
plates (3 × 10⁵ cells/well) were exposed to concentrations
equal to the IC₅₀ for 24 h (70 μM), determined prior
to the experiment. After the cells had been stained with the Annexin
V–FITC and propidium iodide, the percentage of apoptotic cells
was analyzed by flow cytometry (FACS Calibur).

3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), dimethyl sulfoxide (DMSO), M199 and Lectin from Bandeiraea simplicifolia (Griffonia simplicifolia) were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human VEGF₁₆₅ and human basic fibroblast growth factor (bFGF), were obtained from PeproTech Inc. Recombinant human EGF was purchased from ProSpec Company. Matrigel were purchased from BD Bioscience (San Diego, CA). The primary antibodies for VEGF, VEGFR-2, p^Tyr1175-VEGFR-2, p^Tyr951-VEGFR-2, p-p38, p-ERK, p38, ERK, Akt, p-Akt, PI3K p110α, PI3K p85, p-PTEN, p-Colifin, Colifin, cleaved-caspase3/9, cleaved-PARP and all the secondary antibodies were acquired from Cell Signaling Technology (Cell Signaling Technology, Inc, USA). The primary antibodies for GAPDH, β-actin, Bax and Bcl-2 were obtained from Proteintech Group (Proteintech Group, Inc., USA). Annexin V-FITC apoptosis detection kit was purchased from Roche (Indianapolis, IN). Control siRNA (5′-uucuuccgaacgugucacgutt-3′) and siRNA against human VEGFR2 (5′-gcggcuaccaguccggauatt-3′) were synthesized by genepharm. Trypsin and DMEM/F12 were obtained from HyClone, and fetal bovine serum (FBS) was obtained from Gibico. M199 Medium was bought from LIFE. siRNA and all other chemicals were purchased from Sigma Chemical Co. unless otherwise specified.

Apoptosis was measured using the Annexin-V FITC Apoptosis Detection Kit (Roche Applied Science). In brief, cells were fixed in 70% ethanol and resuspended in staining solutions containing fluorescein isothiocyanate (FITC)-conjugated Annexin-V and propidium iodide (PI). Cells were analyzed by a flow cytometer (BD FACSCanto II, Becton Dickinson, USA).