Cell apoptosis was assessed using an Annexin-V/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology). Briefly, cells (5x104 cells per well) were plated in 6-well plates overnight. On the following day, cells were transfected with the plasmids or mimics as aforementioned. Subsequently, the cells were collected, centrifuged with low temperature at high speed (1,000 x g at 4˚C for 5 min) and resuspended in 100 µl of FITC-binding buffer (Beyotime Institute of Biotechnology). Subsequently, ~5 µl ready-to-use Annexin V-FITC (BD Bioscience) and 5 µl PI were added into the buffer. The cells were incubated for 30 min at room temperature in the dark. Annexin V-FITC and PI fluorescence were assessed using a BD FACSCalibur flow cytometer (BD Biosciences). CellQuest software (version 5.1; BD Biosciences) was used to analyze flow cytometry data.
Annexin 5 pi apoptosis detection kit
Manufactured by Beyotime
Sourced in China
The Annexin V/PI apoptosis detection kit is a laboratory instrument used to detect and quantify cell apoptosis. It is based on the binding of Annexin V to phosphatidylserine, a marker for early apoptosis, and propidium iodide (PI) to detect late apoptotic or necrotic cells.
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Lab products found in correlation
Facscalibur flow cytometer, by BD (5 mentions)
Cellquest software, by BD (4 mentions)
Annexin 5 fitc, by BD (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Flow cytometry analysis, by Beckman Coulter (1 mentions)
Laser confocal microscope, by Leica (1 mentions)
Cell counting kit 8 (cck8), by Solarbio (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Beyotime (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Cellquest version 5, by BD (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Facsaria 3, by BD (1 mentions)
Terminal deoxynucleotidyl transferase mediated dutp nick end labeling tunel kit, by Beyotime (1 mentions)
Accuri c6, by BD (1 mentions)
Gallios flow cytometry, by Beckman Coulter (1 mentions)
Docetaxel, by Merck Group (1 mentions)
Mtt kit, by Merck Group (1 mentions)
Lipofectamine 2000 transfection kit, by Thermo Fisher Scientific (1 mentions)
Protein extraction kit, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Zhejiang Tianhang Biotechnology (1 mentions)
32 protocols using annexin 5 pi apoptosis detection kit
1
Annexin-V/PI Apoptosis Detection Assay
2
Cell Viability, Proliferation, and Apoptosis Assays
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For CCK8 analysis, 8000 cells were seeded into each well of a 96-well plate with fresh culture medium. Cell Counting Kit-8 (Solarbio, # CA1210) was used to detect cell viability after 48 h of transfection treatment, and the microplates were incubated at 37 °C. After 4 h, absorbance was recorded at 450 nm using a microplate reader (ThermoFisher) and the relative viability calculated using untreated cells as control (100%). For EdU (5-Ethynyl-2′-deoxyuridine) assay, EdU (Beyotime, # C0071S) was added to the six-well plate and incubated the cells in the logarithmic growth phase for 3 h according to the provided instruction. The cells were washed twice with PBS for 5 min, and then fixed with 4% paraformaldehyde for 30 min. The cells were washed twice with PBS for 5 min each time and infiltrated with 0.3% TritonX-100 in PBS before being stained with the reaction solution. The images were captured with a Leica laser confocal microscope. For the apoptosis assay, cells were incubated staining using Annexin V-PI Apoptosis Detection Kit (Beyotime, #C1065M) according to the provided instruction, followed by flow cytometry analysis (Beckman).
3
Apoptosis Analysis of BMECs
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After being co-cultured for 3 days, BMECs were collected for an analysis of apoptosis that was performed using an Annexin V/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology). In brief, the cells were re-suspended and then incubated with 10 μl of Annexin-V and 10 μl of PI for 15 min in the dark. Subsequently, the apoptotic rate was determined with a FACSAria flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
4
Quantifying Apoptosis via Annexin V/PI Assay
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To analyze cell apoptosis, Annexin V/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. KGN cells were collected by trypsinization through centrifugation (1,000 x g, 5 min, 4˚C), washed with PBS and then resuspended in 1X binding buffer at a density of 1x106 cells/ml. Subsequently, 100 µl cell suspension was added into a 5 ml tube, which was incubated with 5 µl Annexin V-FITC and PI at room temperature for 20 min, according to the manufacturers' protocols. The stained cells were analyzed using a BD FACSCalibur flow cytometer (BD Biosciences) and apoptotic rate (the percentage of early + late apoptotic cells) was calculated. Data were analyzed using CellQuest™ software (version 5.1; BD Biosciences). All the experiments were performed ≥3 times.
5
Quantification of Apoptosis by Flow Cytometry
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Cell apoptosis was assessed using the Annexin-V/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, cells were seeded (2×105 cells/well) into 6-well plates and cultured at 37°C overnight. Following transfection, cells were harvested, centrifuged at 1,000 × g at 4°C for 5 min), and the cell pellet was resuspended in 100 µl FITC-binding buffer. Subsequently, the cell suspension was incubated with 5 µl ready-to-use Annexin V-FITC (BD Bioscience) and 5 µl PI at room temperature in the dark for 30 min. Cell apoptosis (late or early + late apoptosis) was assessed via flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using CellQuest™ version 5.1 software (BD Biosciences).
6
Apoptosis Analysis by Annexin V/PI and TUNEL
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Apoptosis assay was performed using an annexin V/PI apoptosis detection kit (Beyotime, Shanghai, China). The cells were harvested for annexin V/PI staining according to the manufacturer’s instructions and analyzed using flow cytometry (BD FACSAria III, BD Biosciences, Franklin Lakes, NJ, USA). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) kit (Beyotime, Shanghai, China) was applied to further determine cell apoptosis according to the manufacturer’s protocol. GMECs were fixed with 4% paraformaldehyde for 30 min and incubated in 0.3% Triton X-100 for 5 min at room temperature, followed by treatment with TUNEL reaction mixture for 60 min at 37 ℃. After three washes with PBS, the images were acquired using fluorescence microscope (Olympus, Tokyo, Japan).
7
Pazopanib-Induced Apoptosis Evaluation
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Apoptotic cells were evaluated following pazopanib treatment via annexin V/PI apoptosis detection kit from Beyotime. The NCI‐H446 or NCI‐H82 cells were treated with different concentrations of pazopanib. After 24 h, the staining buffer (200 μl) was used to resuspend the cells at a density of 1 × 105. The annexin V and PI (5 μl, respectively) were mixed in the dark for 30 min at room temperature. Subsequently, a BD Accuri C6 (BD Biosciences) flow cytometer was used to detect the apoptotic cells. FlowJo 10.0 software (BD Biosciences) was used to analyze the data.
8
Apoptosis Assay of U2OS Cells
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U2OS cells were transfected with control-siRNA, C5orf66-AS1-siRNA, C5orf66-AS1-siRNA+inhibitor control, or C5orf66-AS1- siRNA+miR-149-5p inhibitor at 37°C for 48 h, and U2OS cell apoptosis was detected using a Annexin-V/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology). Briefly, 200 µl Annexin V-FITC and 10 µl PI were added into the U2OS cell (106 cells) suspension for 30 min at 37°C in the dark according to the manufacturer's protocol. The apoptotic cell rate was determined using a flow cytometer (BD Biosciences) and quantified using FlowJo software (version 7.2.4; FlowJo LLC).
9
Apoptosis Analysis via Annexin V/PI
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Apoptosis was analyzed using the Annexin V/PI apoptosis detection kit (Beyotime, China) according to the manufacturer’s instructions. Briefly, 3 × 105 cells were resuspended in 400 μL of Annexin V binding buffer, and stained with 5 μl Annexin V in the dark for 15 min. Then, 10 μL PI was added and incubated for another 5 min. Stained cells were immediately measured by Gallios flow cytometry (Beckman Coulter, CA, USA).
10
Investigating Docetaxel Resistance in Gastric Cancer
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Human gastric cancer SGC7901 cells and drug-resistant SGC7901/Docetaxel cells were kindly donated by the Basic Medical College of Zhengzhou University. Thirty female nude mice 4 weeks old, weighing 10–15 g, were provided by the Animal Experimental Center of Zhengzhou University. RPMI-1640 culture medium and fetal bovine serum (FBS) were purchased from Zhejiang Tianhang Biotechnology Co., Ltd. (Hangzhou, China). Lipofectamine 2000 Transfection kit was purchased from Invitrogen Co. (Carlsbad, CA, USA). Docetaxel and MTT kits were provided by Sigma-Aldrich Co. (St. Louis, MO, USA). Annexin V/PI Apoptosis Detection kit and Protein Extraction kit were from Beyotime Biotechnology (Shanghai, China). STMN-1 (MDR1 or ERCC1 or caspase 3) mouse anti-human monoclonal antibody and rabbit anti-mouse antibody second antibody were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
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