Cells were treated with HOCl (0–250 μM; in PBS, 1 h) followed by 23 h post-exposure culture in regular medium. Cell viability was then determined using flow cytometric analysis of annexinV (AV)-propidium iodide (PI) stained cells using an apoptosis detection kit (APO-AF, Sigma, St. Louis, MO) according to the manufacturer's specifications as published before [38 (link)].
Apoaf
Manufactured by Merck Group
Sourced in United States
The APOAF is a piece of laboratory equipment manufactured by Merck Group. It serves as a core function for analytical purposes, though its specific intended use is not provided in this concise and unbiased description.
Automatically generated - may contain errors
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27 protocols using apoaf
1
HOCl Treatment and Cell Viability
2
Apoptosis and Necrosis Quantification
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Cells were harvested by trypsinization, pooled with the culture medium containing floating cells, collected by centrifugation, and incubated with the Annexin V-FITC conjugate and propidium iodide (PI) for 10 minutes using an apoptosis detection kit (APOAF, Sigma-Aldrich), then analyzed by flow cytometry. The percentage of apoptosis and necrosis was evaluated by plotting cell staining by Annexin V (x-axis) vs PI (y-axis). The upper left quadrant (PI positive and Annexin V negative, Q1) represents necrotic/non-viable cells; the upper right quadrant (PI positive and Annexin V positive, Q2) represents necrotic/late apoptotic cells; the lower left quadrant (PI and Annexin V negative, Q3) represents live cells; and the lower right quadrant (PI negative and Annexin V positive, Q4) represents early apoptosis.
3
Annexin V/PI Assay for Cell Viability
4
Evaluating Apoptosis in Cancer Cells
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After D2O (up to 90%; up to 6 days) treatment, viability of melanoma and pancreatic cancer cells was assessed by annexinV-FITC/propidium iodide (PI) dual staining followed by flow cytometric analysis as published before using an apoptosis detection kit according to the manufacturer’s specifications (APOAF, Sigma Aldrich, St. Louis, MO, USA) [48 (link),49 (link)].
5
Flow Cytometric Apoptosis Analysis
6
Methylglyoxal and Serum Starvation Impact on Cell Viability
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After treatment [methylglyoxal in medium (500 µm) or serum starvation in Hank’s Balanced salt solution (HBSS) containing 1 g/L glucose (24 h continuous exposure)], viability of A375-GLO1_WT and KO clones was assessed by annexinV-FITC/propidium iodide (PI) dual staining followed by flow cytometric analysis as published before [23 (link),45 (link),46 (link)]. Cells (100,000) were seeded on 35 mm dishes and received treatment 24 h later. Cells were then harvested 24 h later and stained using an apoptosis detection kit according to the manufacturer’s specifications (APOAF, Sigma Aldrich, St. Louis, MO, USA). Viable cells are located in the bottom left quadrant (annexinV-FITC−/PI−), whereas early apoptotic and late apoptotic/necrotic cells are located in the bottom right (annexinV-FITC+/PI−) and top right quadrants (annexinV-FITC+/PI+), respectively.
7
Annexin V-FITC and PI Apoptosis Assay
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Applying the detection kit (APOAF, Sigma), Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining were used to observe apoptotic cells. After washing with phosphate-buffered saline (PBS), experimental cells were resuspended in binding buffer. Then, 5 μL of Annexin V-FITC and 5 μL of PI (50 μg/mL) were added to the binding buffer for 5 minutes in the dark. The samples were immediately measured using a flow cytometer. Apoptotic cells were expressed as a percentage of the total number of cells stained. All experiments were performed in triplicate and repeated twice to assess the consistency of response.
8
Quantifying Cell Apoptosis by Flow Cytometry
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Cell apoptosis were detected by using double staining with Annexin V-FITC/PI (APOAF, Sigma) according to the manufacturer’s instructions. Briefly, cells were harvested by trypsin digestion and washed twice with precooled PBS. The cell pellets were suspended in 1 × binding buffer (10 mM HEPES/NaOH, 140 mM NaCl and 2.5 mM CaCl2, pH 7.4) at a concentration of 5 × 106 cells ml−1. Then the cells were incubated with AnnexinV-FITC and propidium iodide (PI) for 15 min (22–25 °C) in dark. The stained cells were immediately analysed by a BD Acurri C6 flow cytometry (BD, Franklin Lakes, NJ, USA). All data were analysed with Flowjo 7.6. Each measurement was carried out at least in triplicate.
9
Annexin-V-FITC/PI Cell Viability Assay
10
Apoptosis and Necrosis Induction Assay
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Viability and induction of cell death (early and late apoptosis/necrosis) were examined by annexinV-FITC/propidium iodide (PI) dual staining of cells followed by flow cytometric analysis [38 (link)]. Cells (100,000) were seeded on 35 mm dishes and received drug treatment at different concentrations 24 h later. Cells were harvested 24 h after treatment and cell staining was performed using an apoptosis detection kit according to the manufacturer’s specifications (Sigma Chemical Co, APOAF). Viable cells are located in the bottom left quadrant (annexinV-FITC−/PI−), whereas early apoptotic and late apoptotic/necrotic cells are located in the bottom right (annexinV-FITC+/PI−) and top right quadrants (annexinV-FITC+/PI+), respectively.
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