Apoptosis/ Necrosis Assay Kit (blue, green, red) (ab176749)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
• Assay type: Cell-based • Platform: Flow cytometer, Fluorescence microscope • Assay time: 1 hr • Sample type: Adherent cells, Suspension cells	• 1×10^4/well in flat-bottom 96-well culture plates

Upstream tips
• Assay type: Cell-based • Platform: Flow cytometer, Fluorescence microscope • Assay time: 1 hr • Sample type: Adherent cells, Suspension cells
Protocol tips
• 1×10^4/well in flat-bottom 96-well culture plates

Publication protocol

Cellular senescence was determined by β-gal staining as described previously (Gey and Seeger, 2013). Apoptosis/necrosis was de-tected using the commercial kit (AB176749; Abcam, Cambridge, England) following the manufacturer’s instructions. The Apoptosis/Necrosis Detection Kit is designed to simultaneously monitor apoptotic, necrotic, and healthy cells with a flow cytometer or fluores-cence microscope. In apoptotic cells, PS is transferred to the outer side of the plasma membrane and can be detected by its sensor, Apopxin Green (green fluorescence). The 7-aminoactinomycin D (red fluorescence), a membrane impermeable dye, will label the nucleus of damaged cells, including both late apoptotic and necrotic cells. Live cells can be detected as blue fluorescence by CytoCalcein Violet 450 staining. Cytotoxicity was also examined by propidium iodide permeability assay. After damage induction and inhibitor treatment, propidium iodide and Hoechst 33342 were added into the medium to achieve final concentrations of 0.8 and 0.5 μg/ml, respectively. The cells were then incubated at 37°C for 5–15 min and visualized for the total cells (blue, Hoechst 33342) and dead cells (red, propidium iodide) under a fluorescence microscope.

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Manufacturer protocol

Download the product protocol from Abcam for Apoptosis/ Necrosis Assay Kit (blue, green, red) (ab176749) below.

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