At 24 h after transfection with miRNA- 203a-3p mimics or negative controls, the cells were washed with PBS and fixed with 70% ethanol for >12 h at 4°C. Propidium iodide (PI) staining solution (500 µl) was then added to the centrifuged cells (845 × g, 3 min) at room temperature, followed by incubation for 30 min in the dark at room temperature. Cell apoptosis was analyzed by FACSCalibur flow cytometry (BD Biosciences). The percent of apoptotic cells was obtained from FACSCalibur flow cytometry (BD Biosciences) which was used for further calculation. The Annexin V/PI Apoptosis Detection Kit (Beijing Solarbio Science & Technology, Co., Ltd.) was used to assess apoptosis.
Annexin 5 pi apoptosis detection kit
Manufactured by Solarbio
Sourced in China
The Annexin V/PI Apoptosis Detection Kit is a laboratory reagent used for the detection and quantification of apoptosis in cell samples. It utilizes Annexin V, a protein that binds to phosphatidylserine, and propidium iodide (PI), a DNA-binding dye, to differentiate between viable, early apoptotic, and late apoptotic or necrotic cells.
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6 protocols using annexin 5 pi apoptosis detection kit
1
Quantifying Apoptosis via Flow Cytometry
2
Apoptosis Detection in HepG2 Cells
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Cell apoptosis was determined by the Annexin V/PI apoptosis detection kit (Solarbio). Annexin V can bind to phosphatidylserine on the surfaces of HepG2 apoptotic cells and is detected with a flow cytometer in the FITC channel, as a marker for apoptosis. PI can enter the dying or dead cells and is detected with a flow cytometer in PE channel, as a marker for cell death. The cells were segregated into four quadrants: Annexin V−/PI− (viable cells), Annexin V+/PI− (early apoptotic cells), Annexin V−/PI+ (necrotic cells), and Annexin V+/PI+ (late apoptotic cells). HepG2 cells were digested with trypsinization (Procell), centrifuged at 1000× g rpm for 5 min, and then collected. Subsequently, the cells were washed and resuspended in pre-cooled PBS and centrifuged for recollection. This elution step was repeated twice. Then, the cells were resuspended in 1 × binding buffer and incubated with Annexin V and PI staining solution at room temperature for 5 min. Cellular apoptosis was assessed via flow cytometry (Mindray, Shenzhen, China). Analysis was performed using Flow Jo software version 9.3 (Tree Star, Ashland, OR, USA) [38 (link),39 (link),40 (link)].
3
Annexin-V/PI Apoptosis Detection
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Apoptosis was detected using an Annexin-V/PI apoptosis detection kit (Solarbio Life Sciences, Beijing, China) according to the manufacturer’s instructions. Finally, the percentage of apoptotic cells was assessed by flow cytometry (Beckman).
4
Annexin V-PI Apoptosis Detection
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Apoptotic cells were detected using an annexin V/ PI apoptosis detection kit (CA1020, Solarbio). Briefly, cells were grouped and treated as described above, and then cells were diluted at 10 6 /ml using binding buffer. Cells were incubated with annexin V for 10 min followed by incubation with PI for 5 min. Apoptotic cells were detected using NovoCyte.
5
Evaluating MSC Apoptosis Response
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BMSCs (approximately 106 cells) were cultured with DMEM in a 6-well plate. After incubating overnight for cell adherence, the original DMEM was substituted with the suspension that contained the same concentration of HMS, QHMS, and Ag@QHMS for 72 hours. Subsequently, the medium was discarded and rinsed twice with cold PBS, which was followed by cell digestion with EDTA-free trypsin. It was then resuspended to a cell density of 105 according to the instructions of the annexin V-PI apoptosis detection kit (Solarbio Science & Technology Co., Beijing, China) and then analyzed on an Accuri™ C6 flow cytometer (BD Bioscience, Shanghai, China).
6
Apoptosis and Cell Cycle Analysis
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Annexin V/PI Apoptosis Detection Kit (Solarbio, Beijing, China) was used to evaluate cell Apoptosis rate according to the manufacturer’s instructions. Briefly, cells to be tested were harvested using trypsin without EDTA, washed twice with cold PBS and once with cold binding buffer, then resuspended with cold binding buffer and incubated with Annexin V-FITC and PI staining solution for 15 min at room temperature in the dark. Samples were analyzed by flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Each independent experiment was performed at least three times.
PI staining was used to detect cell cycle. In brief, cells to be tested were collected and washed with cold PBS three times and resuspended with 70% cold ethanol at 4 °C overnight. Then, cells were stained with 50 µg/mL PI containing RNase (100 µg/mL) for 30 min. The results were obtained from cytometry. Each experiment was performed at least three times.
PI staining was used to detect cell cycle. In brief, cells to be tested were collected and washed with cold PBS three times and resuspended with 70% cold ethanol at 4 °C overnight. Then, cells were stained with 50 µg/mL PI containing RNase (100 µg/mL) for 30 min. The results were obtained from cytometry. Each experiment was performed at least three times.
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