RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
• A live-cell real-time assay that measures the exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane during the apoptotic process • Detected by annexin V binding with a simple luminescence signal. • Assay also includes a cell-impermeant, profluorescent DNA dye, which detects necrosis	• 25 × 10^3 cells/well on 96 well plates	• Assay, time-dependent increases in luminescence that occur before increases in fluorescence reflect the apoptotic process. • A significant time delay between the emergences of PS, indicated by Annexin V binding, leading to a luminescence signal and the loss of membrane integrity visualized by fluorescence signal, indicate an apoptotic phenotype leading to secondary necrosis. • Increases in fluorescence or increase in both luminescence and fluorescence concurrently consist of necrosis or other non-apoptotic mechanisms.

Upstream tips
• A live-cell real-time assay that measures the exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane during the apoptotic process • Detected by annexin V binding with a simple luminescence signal. • Assay also includes a cell-impermeant, profluorescent DNA dye, which detects necrosis
Protocol tips
• 25 × 10^3 cells/well on 96 well plates
Downstream tips
• Assay, time-dependent increases in luminescence that occur before increases in fluorescence reflect the apoptotic process. • A significant time delay between the emergences of PS, indicated by Annexin V binding, leading to a luminescence signal and the loss of membrane integrity visualized by fluorescence signal, indicate an apoptotic phenotype leading to secondary necrosis. • Increases in fluorescence or increase in both luminescence and fluorescence concurrently consist of necrosis or other non-apoptotic mechanisms.

Publication protocol

The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay were performed as prescribed by the manufacturer. Briefly HUVECs were seeded with a density of 25 × 103 cells/well on 96 well white plates (96F Nunclon™ Delta White Microwell SI, Thermo Fisher Scientific, Roskilde, Denmark) in 50 μl medium (SCME001, Millipore, Billerica, MA, USA) and incubated at 37 °C in 5% CO2, humidified air for 24 h. Afterwards cells were preincubated for 1 h in the presence or absence of the TRPV4 antagonist GSK2193874. Cells were then treated with different concentrations of the TRPV4 agonists GSK1016790A or 4α-PDD and directly equal volume of detection reagent was added, the plate was covered with an imaging seal (4titude 4ti-0516/96, LabSource, Switzerland) and a kinetic mode (1 read every 30 s for up to 20 h) using a multimode instrument with temperature control was initiated for assay signal detection.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Necrosis HUVEC using RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay from Promega.

Manufacturer protocol

Download the product protocol from Promega for RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Necrosis HUVEC using RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay from Promega. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.