Hoechst 33342

Protocol tips

Upstream tips	Protocol tips	Downstream tips
• Differential Staining for apoptosis and necrosis	• Seed 15,000 cells/well	•Apoptotic and necrotic cells were counted and the percentage number of cells was calculated.

Upstream tips
• Differential Staining for apoptosis and necrosis
Protocol tips
• Seed 15,000 cells/well
Downstream tips
•Apoptotic and necrotic cells were counted and the percentage number of cells was calculated.

Publication protocol

Cells were seeded in 24-well plate (15,000 cells/well) and incubated at 37°C in a humidified atmosphere containing 5% CO2. After 24 h, 90%, 50%, and 10% of EC50 of NSAIDs were added to the wells and incubated for 72 h. Then 3 μL Hoechst 33342 (1 mg/mL; Invitrogen, Paisley, UK) and 1 μL propidium iodide (1 mg/mL, Invitrogen) were added to each well and cells were incubated for 10 min. Images were taken using inverted fluorescent microscope (Olympus IX73). Apoptotic and necrotic cells were counted and the percentage number of cells was calculated.

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Manufacturer protocol

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