A certain dose of ANGPTL4 was used to treat 1 × 105 BMSCs, cultured in 12 well plate with serum‐free DMEM for 24 h, centrifuged at 1500 g for 5 min, and washed twice with 1 × PBS. The cells were fixed with 70% ethanol (Solarbio, Beijing), placed at 20°C for 15 min, centrifuged at 1500 g for 5 min, and washed twice by precooling 1 × PBS. Subsequently, the cells were incubated with 10 μg/μl Dnase‐free RnaseA (Sigma, St. Louis, MO, USA) to remove RNA, and washed twice with precooled 1 × PBS. Finally, after centrifugation at 150 g for 5 min, the cells were incubated with 1 mg/ml iodide (Sigma) in the dark at 4°C for 12 min. Flow cytometry and ModiFit software (Olympus, Tokyo, Japan) were used to quantify the reactive oxygen species (ROS) level and glucose uptake of BMSCs. In addition, according to the instructions, annexin FITC/PI apoptosis detection kit (Beyotime, Nanjing, China) was combined with flow cytometry to detect apoptosis (Dong & Cui, 2017 ). ModiFit software (Olympus, Tokyo, Japan) was performed to analyze the data.
Annexin fitc pi apoptosis detection kit
Manufactured by Beyotime
Sourced in China
The Annexin-FITC/PI Apoptosis Detection Kit is a laboratory product used to detect and quantify apoptosis in cell samples. It contains Annexin V conjugated with fluorescein isothiocyanate (FITC) and propidium iodide (PI) as key components.
Automatically generated - may contain errors
4 protocols using annexin fitc pi apoptosis detection kit
1
ANGPTL4 Modulates BMSC ROS and Apoptosis
2
Annexin V-FITC/PI Apoptosis Assay in Chondrocytes
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Chondrocyte apoptosis was evaluated using an Annexin FITC/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions following transfection and treatment. Chondrocytes were harvested, washed with PBS (Beijing Solarbio Science & Technology Co., Ltd.), resuspended at a concentration of 1.0x106 cells/ml and incubated with 5 µl Annexin V-FITC and 10 µl PI for 10 min at room temperature in the dark. Chondrocyte apoptotic rate was analyzed using a FACScan® flow cytometer (BD Biosciences) for 1 h. The level of apoptotic cells were analyzed using FlowJo 7.6.1. (FlowJo LLC). Apoptosis rate was calculated as the sum of the early apoptosis rate (the lower right quadrant) and the late apoptosis rate (the upper right quadrant).
3
Cell Cycle and Apoptosis Analysis
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A total of 1×105 SW480 or LoVo cells were cultured in 12-well plate by using serum-free DMEM for 24 h, centrifuged at 150 × g for 5 min, and washed twice with pre-cooled 1X PBS. Subsequently, the cells were fixed with 70% ethanol (Solarbio), maintained at 20°C for 15 min, centrifuged at 150 × g for 5 min, and washed twice with pre-chilled 1X PBS. Next, the cells were incubated with 10 µg/µl DNase-free RNaseA (Sigma-Aldrich; Merck KGaA) for 45 min at 37°C to eliminate RNA, and washed twice with pre-chilled 1X PBS. Finally, following centrifuging at 150 × g for 5 min, the cells were incubated with 1 mg/ml iodide (Sigma-Aldrich; Merck KGaA) in the dark at 4°C for 12 min. The cell distribution at each phase of the cell cycle was quantified in a flow cytometer and using ModFit software 3.3 (Verity Software House). In addition, according to the manufacturer's instructions, Annexin-FITC/PI Apoptosis Detection Kit (Beyotime Institute of Biotechnology) was combined with flow cytometry to detect cell apoptosis (19 (link)). ModFit software 3.3 was performed to analyze the data.
4
Cell Cycle Analysis and Apoptosis Assay
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A total of 1 × 105 chondrocytes were cultured in a 12-well plate using serum-free DMEM for 24 h, centrifuged at 1500 g for 5 min and washed with pre-cooled 1 × PBS twice. Subsequently, cells were fixed with 70% ethanol (Solarbio, Beijing, China), placed at 20 °C for 15 min, then centrifuged at 1500 g for 5 min, and washed twice with pre-chilled 1 × PBS. Subsequently, cells were incubated with 10 μg/μL DNase-free RNaseA (Sigma, St. Louis, USA) for 45 min at 37 °C to eliminate RNA, and washed twice with pre-chilled 1 × PBS. Finally, following centrifuging at 150g for 5 min, the cells were incubated with 1 mg/mL iodide (Sigma, St. Louis, USA) in the dark at 4 °C for 12 min. The percentage of cells at each cell cycle phase is quantified in a flow cytometer and analyzed by ModiFit software (Olympus, Tokyo, Japan). Besides, according to instructions, Annexin-FITC/PI Apoptosis Detection Kit (Beyotime, Nanjing, China) was combined with flow cytometry to detect cell apoptosis, according to manufacturer’s instructions [17 (link)]. In addition, modified software (Olympus, Tokyo, Japan) was performed to analyze the data.
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