Quantitative apoptotic cell death by plasma was detected using the Annexin V-propidium iodide (PI) apoptosis detection kit I according to the manufacturer's protocol (BD Biosciences, Bedford, MA, USA), as described previously.12 (link) The cells were treated with various gas device (Ar, N2, and He) NTP for 1, 3, and 5 min and then incubated further for 24 h. The cells were harvested, washed with cold phosphate-buffered saline (PBS), and stained with Annexin V-fluorescein isothiocyanate and PI at room temperature for 15 mins in the dark. The early and late apoptosis were quantified according to the manufacturer's instructions. Apoptosis was detected using a FACS Aria system (BD Biosciences), with the excitation and emission settings of 488 and 530 nm, respectively.
Annexin 5 propidium iodide apoptosis detection kit 1
Manufactured by BD
Sourced in United States
The Annexin V-propidium iodide (PI) apoptosis detection kit is a laboratory tool used to identify and quantify cells undergoing apoptosis, a programmed cell death process. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, and propidium iodide, a fluorescent dye that stains DNA, to differentiate between viable, apoptotic, and necrotic cells.
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7 protocols using annexin 5 propidium iodide apoptosis detection kit 1
1
Apoptosis Detection in NTP-Treated Cells
2
Quantifying Apoptosis by Annexin V-PI
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Quantitative apoptotic cell death by NTS was detected using the Annexin V-propidium iodide (PI) apoptosis detection kit I (BD Biosciences) according to the manufacturer’s instructions. Cells were treated with NTS for 24 hours. The cells were then harvested, washed with cold PBS, and stained with Annexin V-fluorescein isothiocyanate and PI at room temperature for 15 minutes in the dark. Apoptosis was detected using a BD FACSAria III cell sorter (BD Biosciences).
3
Quantifying Apoptosis by Flow Cytometry
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48 h after transient transfection, according to the annexin V-propidium iodide (PI) apoptosis detection kit I (BD Pharmingen, CA, USA) protocol, cells were harvested and re-suspended with 500 μL of binding buffer. The cell suspension was incubated with 5 μL of annexin V-fluorescein isothiocyanate (FITC) and 5 μL of PI buffer at room temperature for 20 min. The stained cells were analyzed on a flow cytometer (BD Biosciences, NJ, USA) and data were analyzed via FlowJo (FlowJo, Ashland, OR, USA). Apoptotic cells were quantified by the apoptosis ratio. The experiment was repeated three times.
4
Quantifying Apoptosis via Annexin V/PI
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An Annexin V/propidium iodide (PI) apoptosis detection kit I (BD Biosciences) was used to measure apoptosis. The cells from each group were collected, washed twice with ice-cold PBS and re-suspended in 200 µl binding buffer containing 10 µl Annexin V and 10 µl PI. Fluorescence intensity was measured by flow cytometry (BD Biosciences). All the samples were assayed in triplicate.
5
Quantitative Apoptosis Assessment in HaCaT Cells
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Quantitative apoptotic cell death of HaCaT cells and fibroblasts was detected using the Annexin V-propidium iodide (PI) Apoptosis Detection Kit I according to the manufacturer’s protocol (BD Biosciences, Bedford, MA, USA). Briefly, the cells were treated with PAW and incubated for 24 h. The cells were harvested, washed with cold PBS, and stained with Annexin V-fluorescein isothiocyanate and PI at room temperature for 15 min in the dark. Early and late apoptosis were quantified according to the manufacturer’s instructions. Apoptosis was detected using a FACSAria System (BD Biosciences) with excitation and emission wavelengths of 488 and 530 nm, respectively. The experiment on H2O2-treated cells as positive control was also performed in the same manner.
6
Quantitative Analysis of CDDP-Induced Apoptosis
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Quantitative apoptotic cell death by CDDP was detected using the Annexin V-propidium iodide (PI) apoptosis detection kit I (BD Biosciences, Bedford, MA, USA) according to the manufacturer's instructions. TPC1 cells were treated with CDDP for 16 hours. The cells were harvested, washed with cold phosphate-buffered saline (PBS), and stained with Annexin V-fluorescein isothiocyanate and PI at room temperature for 15 min in the dark. The early and late apoptosis were quantified according to the manufacturer's instructions. Apoptosis was detected using a FACS Aria system (BD Biosciences), with the excitation and emission settings of 488 and 530 nm, respectively.
7
Cell Proliferation and Apoptosis Assays
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Cell proliferation was measured using the WST-1 Colorimetric Assay (Roche). Briefly, after the treatments, 10 μL/well of cell proliferation detection reagent, WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitro- phenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate), was added into 96-well microplate and incubated for additional 3 h at 37°C, 5% CO2 conditions. The absorbance of samples was then measured by using a microplate reader at 450 nm. The flow cytometry was used for the quantitative assessment of apoptosis with the fluorescein isothiocyanate–Annexin V/propidium iodide (PI) Apoptosis Detection Kit I (BD Pharmingen) and analyzed on a FACS Calibur 4-color flow cytometer (BD Bioscience).
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