Annexin 5 fluoresceine isothiocyanate fitc apoptosis detection kit

Ivalin (>98%) was isolated from Carpesium divaricatum and identified by ESI-MS, ¹H, and ¹³C NMR data [16 (link)]. Compound were dissolved at 10 μM in dimethyl sulfoxide (DMSO) as a stock solution and diluted to desired concentrations according to the research requirement. 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Antibodies against phosphor-Cdc2, Cdc2, Cdc25A, Cyclin B1, Bcl-2, and Bax were purchased from Cell Signaling Technology (CST, Inc., Beverly, MA, USA). Tubulin antibody was purchased from Proteintech Group, Inc. (Chicago, IL, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Abcam, Inc. (Cambridge, MA, USA). We bought the Annexin V–fluoresceine isothiocyanate (FITC) Apoptosis Detection Kit from BD Biosciences (San Jose, CA, USA). Propidium iodide (PI) and RIPA lysis buffer were obtained from the Beyotime Institute of Biotechnology (Shanghai, China). Vincristine (VCR) was purchased from Shenzhen Main Luck Pharmaceuticals Inc. (Shenzhen, China).

Fluorescent microscopy, flow cytometric analysis of propidium iodide-stained nuclei and DNA fragmentation assay were performed as described [13] (link). Apoptosis was also determined by translocation of phosphatidylserine to the cell surface using the annexin V-fluoresceine isothiocyanate (FITC) apoptosis detection kit (BD Pharmingen) according to the manufacturer's protocol.

To study changes in the cell DNA content, histogram measurements of hypodiploid DNA formation was performed by flow cytometry using a BD FACSVerse™ cytometer (BD Biosciences, San Jose, CA). Histograms were analysed with the Flowing Software™. Cells were collected and centrifuged at 500 × g, washed with PBS and resuspended in 50 μL of PBS. Following dropwise addition of 1 ml of ice-cold 75% ethanol, fixed cells were stored at −20 °C for 1 h. Samples were then centrifuged at 500 × g and washed with PBS before resuspension in 1 mL of PBS containing 50 μg mL⁻¹ propidium iodide and 100 μg mL⁻¹ RNase A and incubation for 1 h at 37 °C in the dark. The percentage of cells with decreased DNA staining, composed of apoptotic cells resulting from either fragmentation or decreased chromatin, was determined of a minimum of 10,000 cells per experimental condition. Cell debris was excluded from analysis by selective gating based on anterior and right angle scattering. Apoptosis was also determined by translocation of phosphatidylserine to the cell surface using the annexin V-fluoresceine isothiocyanate (FITC) apoptosis detection kit (BD Pharmingen) according to the manufacturer’s protocol.