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	• 5 X 10^5 cells were seeded in 6-well plates for 24h

Upstream tips
• Assay type: Cell-based • Platform: Flow cytometer, Fluorescence microscope • Assay time: 1 hr • Sample type: Adherent cells, Suspension cells
Protocol tips
• 5 X 10^5 cells were seeded in 6-well plates for 24h

Publication protocol

Approximately 5 X 10^5 cells were seeded in 6-well plates for 24 h. Then, cells were treated with an IC50 concentration of D14 and C22 compounds and vehicle for 24 h. Cells were harvested with 0.25% trypsin, washed with phosphate-buffered saline (PBS), and collected together by centrifugation. Apoptosis was determined using the Apoptosis/Necrosis Detection kit (Abcam, catalog No. ab176749, Cambridge, England) according to the manufacturer’s instructions and analyzed by flow cytometry using a FACSCalibur instrument (BD Biosciences), followed by data analysis using FlowJo software (Tree Star Inc). All experiments were performed in triplicate. Proteome Profiler Apoptosis Array (R&D Systems: ARY009) was used to evaluate the activity of D14 and C22 compounds on MIA-PaCa-2 cancer cells to determine the signaling pathways associated with cell death via Kras4B inhibition, which were done following the manufacturer’s instructions.

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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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