Annexin 5 fitc apoptosis detection kit

Cell proliferation was evaluated using a CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS assay; Promega, Madison, WI, USA). The absorbance was measured at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Cell Cycle Detection Kit (Keygentec, Nanjing, China) was used to assessed the cell cycle. An Annexin V-FITC Apoptosis Detection Kit (Keygentec, Nanjing, China) was used to assessed cell apoptosis. The percentages of the cell population in different phases and cell apoptosis were assessed with flow cytometry (BD Biosciences, San Jose, CA, USA). All experiments were repeated in independent triplicate.

An Annexin V-FITC Apoptosis Detection Kit (Keygentec, China) was applied to determine the apoptosis rates following the manufacturer’s instructions. Briefly, Collected chondrocytes were suspended in binding buffer in a concentration of 1 × 10⁵ cells/ml and were stained with 5 μl Annexin V-FITC and 10 μl PI for 15 min in the darkroom. The results were analyzed using a flow cytometer (Becton Dickinson, United States).

After treatment, the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay kit (MTS assay; Promega, Madison, WI, USA) was used to evaluate cell proliferation. Cells were double-stained using the Annexin V-FITC Apoptosis Detection Kit (Keygentec, Nanjing, China), following the manufacturer’s instructions. The percentage of apoptotic cells was assessed by NovoCyte 4025 flow cytometry and NovoExpress software analysis (ACEA Biosciences, Agilent, Hangzhou, China). The original images of apoptosis were shown in Supplementary 4_flow apoptosis pictures.

Cells cultured in 6-well plates were treated with phloretin (0, 20, 50, and 100 μM) for 24 h and then harvested for flow cytometry analysis (cell apoptosis assay) by using an Annexin V-FITC Apoptosis Detection Kit (Keygentec, Nanjing, China) according to the instruction of manufacturer. Flow cytometry analysis for cell apoptosis was performed using the EPICS Elite ESP high-performance cell sorter (Coulter Electronics, Ltd., England, UK) and analyzed by ModFit LT (version 2.0; Verity Software), and a minimum of 30,000 events were collected for each sample.
For DAPI staining assay, cells cultured in 12-well plates were incubated with phloretin (0, 20, 50, and 100 μM) for 24 h. Cells were briefly washed with 1× PBS and fixed in 4% formaldehyde for 15 min, and then washed three times with 1× PBS and permeabilized in 0.2% Triton X-100 for 15 min. Finally, cells were stained with DAPI (1 μg/ml) at 37°C for 30 min in the dark and then observed and photographed by fluorescence microscopy (Nikon, IX-71, Japan).

Cell proliferation and apoptosis analyses were performed at 48 h after transfection. Cell proliferation was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, USA), whereas apoptotic cells were measured using an Annexin V-FITC Apoptosis Detection Kit (Keygentec, China) and a flow cytometer (BD Biosciences), following the manufacturers’ recommended protocols. All experiments were performed in triplicate.