Annexin 5 fitc pi double staining kit

An Annexin V-FITC/PI double-staining kit (KeyGen Biotech, China) was used to detect the apoptosis rate in vitro. In brief, the collected cells were washed twice using 4 °C PBS and then resuspended in 500 μl of binding buffer. Next, 5 μl of FITC and 5 μl of PI were added to the buffer and incubated with MC3T3-E1 cells in the dark at room temperature for 15 min. Finally, all the collected cells were analyzed by FACSCalibur flow cytometer (Becton Dickinson, USA). Annexin V-FITC-positive/PI-negative and Annexin V-FITC-positive/PI-positive cells were considered to be apoptotic cells.
A TUNEL staining assay was used to explore apoptotic osteoblasts in the tibia bone tissue of C57BL/6 mice. Briefly, all tibias were removed, fixed in 4% paraformaldehyde, decalcified with 10% EDTA for 3 weeks, embedded in paraffin, and sliced into 5-μm-thick transverse sections following the standard method. Apoptotic cells in bone tissue were assessed with the deoxynucleotidyl transferase-mediated nick end labeling assay (TUNEL assay, Roche, Mannheim, Germany). Apoptotic osteoblasts were visualized under ZEISS LSM 880 (Carl Zeiss, Germany).

For apoptosis analysis, cells were harvested, washed with PBS and binding buffer, then stained with Annexin V-FITC and PI using Annexin V-FITC/PI double staining kit (KeyGen Biotech) for 30 min. Apoptotic cells were detected by flow cytometry using BD Calibur. For cell cycle, cells were suspended in pre-cold 70% ethanol, then washed with PBS, incubated with RNase A, and stained with PI using Cell cycle detection kit (KeyGen Biotech). Cell cycle proportion was measured using BD Calibur.

1,6-Hexanediamine (92%), N,N-bis(3-aminopropyl)methylamine (98%), chloroform (99%), sodium hydroxide (50 wt% aqueous solution) and benzyltriethylammonium chloride (TEBAC) were purchased from Wanqing Chemical Instrument Co., Ltd (Nanjing, P.R.C.). Paclitaxel, oxaliplatin, cisplatin (DDP), verapamil (VRP) and z-VAD-FMK were obtained from Selleck Chemicals (Houston, USA). Annexin V-FITC/PI double-staining kit was provided by KeyGen (Nanjing, P.R.C.). N-acetyl-L-cysteine (NAC), hemoglobin, Hoechst 33342, crystal violet, 4’,6-diamidino-2-phenylindole (DAPI), 3-amino-4-aminomethyl-2’,7’-difluorescein diacetate (DAF-FM DA) and 2’,7’-dichlorofluorescein (DCFH-DA) were provided by Beyotime Biotechnology (Nanjing, P.R.C.). Propidium iodide (PI), calf thymus DNA (ct-DNA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and ethidium bromide (EB) were purchased from Sigma-Aldrich (St. Louis, USA). γ-H2AX (phospho S139) antibody was obtained from Abcam (Cambridge Science Park, Cambridge, UK). Primary antibodies against specific for cleaved caspase-3, cleaved caspase-9, Bax, poly (ADP-ribose) polymerase (PARP), β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology (Boston, USA).

An Annexin V-FITC/PI double-staining kit (KeyGen Biotech, China) was used to detect apoptosis of neurons and astrocytes in the OGD + GFP MSC and OGD + siIL-6 MSC groups according to the manufacturer’s instructions. Briefly, after treatment, the cells in the two groups were washed three times with PBS and then incubated in 5 μl of FITC-conjugated Annexin V and 5 μl of PI in 100 μl of binding buffer for 5 min at room temperature. Apoptotic changes to neurons and astrocytes were observed under an inverted fluorescence microscope (Nikon, Japan) and photographed using a digital camera (Nikon, Japan).

Apoptosis rate of PEDV-infected Vero cells was assessed with Annexin V-FITC/PI double staining kit (KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. The cells were seeded into 6-well plates and then stained with Annexin V-FITC and PI in dark. Cells were analyzed by flow cytometry (Ex = 488 nm, Em = 525 nm). Cell-Quest software (Becton Dickinson, San Jose, CA, USA) was employed to analyze the data.

Apoptosis of PC12 cells in the control, OGD and OGD + MSCs groups was detected using an annexin V-FITC/PI double-staining kit (KeyGen BioTECH) according to the manufacturer’s recommendations. Briefly, after treatment, the cells in the three groups were washed twice with PBS buffer and then incubated with 5 μl of FITC-conjugated annexin V and 5 μl of PI in 500 μl of binding buffer for 5 min at room temperature. Apoptotic changes in PC12 cells with different treatments were observed under an inverted fluorescence microscope (Nikon, Japan) and photographed using a digital camera.