Ebioscience annexin 5 apoptosis detection kit pe

At various times after treatment with XN, DXN, or TXN, adherent and floating cells were collected for analysis. Apoptosis was assessed using flow cytometry-based annexin V (Invitrogen™. eBioscience™ Annexin V Apoptosis Detection Kit PE, Thermo Fisher, Waltham, MA, USA) and a multicaspase assay kit (MilliporeSigma, Burlington, MA, USA). The annexin V assay is based on measurement of externalization of phosphatidyl serine, a common characteristic of cells undergoing apoptosis. The multicaspase assay is based on measurement of caspase enzymes activated during apoptosis. Cells were trypsinized, washed in Dulbecco’s Phosphate Buffered Saline (D-PBS), and stained with Annexin V, sulforhodamine-valyl-alanyl-aspartyl-fluoromethyl-ketone (SR-VAD-FMK), and 7-amino-actinomycin D (7AAD) according to the manufacturer’s instructions. Cell populations were quantified using a Guava personal cytometer (Guava Technologies, Burlingame, CA, USA).

Cell death was measured after treatment using the Invitrogen™ eBioscience™ Annexin V Apoptosis Detection Kit PE (ThermoFisher, Dublin, Ireland) according to the manufacturer’s instructions. Briefly, after 72 h exposure time, cells grown in a 24-well plate were harvested, centrifuged at 500× g for 5 min and washed with PBS. After a second wash with Binding Buffer, cells were resuspended in 100 μL of Binding Buffer and incubated for 15 min at room temperature with 1 μL of fluorochrome-conjugated Annexin V-PE. After washing with Binding Buffer, cells were incubated with 1 μL 7-AAD Viability Staining Solution. Annexin V-PE and 7AAD fluorescence was then measured using the CytoFLEX Flow Cytometer (Beckman Coulter Inc, Indianapolis, IN, USA) with a blue laser (488 nm) using FL-2 and FL-3 detectors. Results are expressed in the percentage of early apoptotic, late apoptotic/necrotic, necrotic and viable cells.