FITC Annexin V Apoptosis Detection Kit I

Protocol tips

Upstream tips	Protocol tips	Downstream tips
• Cells were washed with PBS, centrifuged and stained with Annexin V-FITC for 20 min at room temperature in the dark and subsequently stained with propidium iodide (PI) for 5 min before the measurement.	• A total number of 10,000 cells per sample were analyzed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Fluorescence was detected via a 530/30 nm band-pass filter (FL-1; Annexin V—FITC) and a 670 nm long-pass filter (FL-3; PI). Raw files were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).	• For the measurement of the viability and phosphatidylserine externalization as a marker of apoptosis, a BD Pharmingen FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) was used according to the manufacturer’s instructions.

Upstream tips
• Cells were washed with PBS, centrifuged and stained with Annexin V-FITC for 20 min at room temperature in the dark and subsequently stained with propidium iodide (PI) for 5 min before the measurement.
Protocol tips
• A total number of 10,000 cells per sample were analyzed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Fluorescence was detected via a 530/30 nm band-pass filter (FL-1; Annexin V—FITC) and a 670 nm long-pass filter (FL-3; PI). Raw files were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).
Downstream tips
• For the measurement of the viability and phosphatidylserine externalization as a marker of apoptosis, a BD Pharmingen FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) was used according to the manufacturer’s instructions.

Publication protocol

Apoptosis and necrosis were determined as reported previously (12). 100,000 cells were cultured at 37°C in 6-well plates; the next day, they were treated with 3G (20 µM) at 37°C for 24 h. Detection of apoptosis was performed using the Annexin V/Dead Cell Apoptosis kit (cat. no. V13242, Molecular Probes; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. Cells were resuspended in Annexin-binding buffer and incubated at 25°C with Annexin V-fluorescein isothiocyanate (FITC; 5 µl) and propidium iodide (PI; 1 µl) for 15 min. Data acquisition and analysis were performed using CellQuest Pro Version 6.0 BD FACSCALIBUR (BD Biosciences) and analysis were performed, wherein fluorescence emission was measured at 530 nm (FL1 channel) for FITC Annexin V and >575 nm (FL3 Channel) for PI; 10,000 events were used for each test.

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