Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide (PI)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 12 × 104 cells/well were platted in 6 well plate. - Cells were exposed to to MPP+ (1000 μM), α-mangostin (10 μM), or the combination of MPP+ (1000 μM) and α-mangostin (10 μM) for 24 hours.	- Cells were incubated withFITC annexin-V and PI at room temperature for 15 minutes

Upstream tips
- 12 × 104 cells/well were platted in 6 well plate. - Cells were exposed to to MPP+ (1000 μM), α-mangostin (10 μM), or the combination of MPP+ (1000 μM) and α-mangostin (10 μM) for 24 hours.
Protocol tips
- Cells were incubated withFITC annexin-V and PI at room temperature for 15 minutes

Publication protocol

SH-SY5Y cells were seeded onto 6-well plates at a density of 12 × 104 cells/well in 2 mL of medium and then incubated at 37°C under 5% CO2 in a humidified incubator for 24 hours. After exposure to MPP+ (1000 μM), α-mangostin (10 μM), or the combination of MPP+ (1000 μM) and α-mangostin (10 μM) for 24 hours, cells were trypsinized and centrifuged at 2,500 rpm, 4°C for 5 minutes. The pellet was washed twice with cold PBS and resuspended at a concentration 1 × 106 cells/mL. Apoptotic cells were detected using an FITC annexin-V/dead cell apoptosis kit (Molecular Probes, Eugene, OR, USA). Cells were incubated with 5 μL of FITC annexin-V and 1 μL of the 100 μg/mL propidium iodide (PI) at room temperature for 15 minutes. The apoptotic cells were analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA), measuring the fluorescence emission at 530 nm (FL1) and >575 nm (FL3). Both early apoptotic (annexin-V-positive and PI-negative) and late apoptotic (annexin-V-positive and PI-positive) cells were included in cell death determinations.

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