Cells treated with the indicated doses of chrysosplenol D (0, 25, 50, and 100 µM) were collected and resuspended in phosphate-buffered saline (PBS) with 2% bovine serum albumin (BSA). Subsequently, cells were incubated with annexin V–fluorescein isothiocyanate solution and propidium iodide (PI) solution (BD Biosciences, San Jose, CA, USA) in the dark. The percentage of apoptotic cells was measured using a BD Accuri C6 Plus flow cytometer (BD Biosciences), and data were analyzed using BD CSampler Plus software (BD Biosciences).
Annexin 5 fluorescein isothiocyanate solution
Manufactured by BD
Sourced in United States
Annexin V–fluorescein isothiocyanate solution is a laboratory reagent used to detect and quantify apoptosis, or programmed cell death, in cell samples. It is a fluorescent-labeled derivative of the Annexin V protein, which binds to phosphatidylserine, a component of the cell membrane that is exposed during apoptosis. The fluorescent label, fluorescein isothiocyanate, allows the detection and analysis of apoptotic cells using flow cytometry or fluorescence microscopy.
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3 protocols using annexin 5 fluorescein isothiocyanate solution
1
Chrysosplenol D Induces Apoptosis
2
Picrasidine I Induces Apoptosis
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NPC-039 and NPC-BM cells were seeded into a 6-well plate at a density of 3 × 105 cells per well and exposed to the indicated dose of picrasidine I (0, 10, 20, 40 µM) for 24 h. After 24 h, cells were collected and incubated with annexin V–fluorescein isothiocyanate solution and PI solution (BD Biosciences) for 20 min in the dark at room temperature. The percentage of apoptotic cells was analyzed using the BD CSampler Plus software (version 1.0; BD Biosciences).
3
Apoptosis Quantification in Prostate Cancer
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Cells (2×105) were plated in 6-well dishes. Following attachment, cells were treated with JQ1 (10, 20, 40 µM for DU145, 100, 200, 400 nM for LNCAP) for 48 h or transfected with shBRD4 as aforementioned. Cell suspensions were harvested via 0.25% trypsinization for 45 sec at 37°C without EDTA, washed with PBS, and resuspended in 250 ml binding buffer [Hangzhou Multisciences (Lianke) Biotech Co., Ltd., Hangzhou, China]. Cell suspensions were stained with 5 µl PI and 5 µl Annexin V-fluorescein isothiocyanate solution (BD Biosciences) and incubated at room temperature for 15 min in the dark. Finally, a flow cytometer (BD FACSCalibur; BD Biosciences) was used to quantify apoptotic cells. CellQuest 3.0 software (BD Biosciences) was used to analyze the data. The rate of apoptosis was obtained from three repeats, and determined with GraphPad Prism 6.
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