Apoptosis was detected by an Annexin V-APC/7AAD Apoptosis Detection Kit (GA1023-KGA1026, Keygentec, Nanjing, China) followed by flow cytometry analysis. Briefly, cells were treated or not with the indicated drug concentration and time, washed with PBS, stained with Annexin V-APC/7AAD and detected using flow cytometry (CytoFLEX flow cytometer, Beckman Coulter, Inc.). The apoptosis rate of stained cells was counted in APC/PC5.5. FACS sequential gating strategies of apoptosis analysis was showed in Supplementary Fig. 21A . Cell % in per group are presented as mean ± SEM. Significance was analyzed by equal variance two-tailed t test. *P < 0.05, **P < 0.01, *** P < 0.001.
Annexin 5 apc 7 aad apoptosis detection kit
Manufactured by Keygen Biotech
Sourced in China, United States
The Annexin V-APC/7-AAD Apoptosis Detection Kit is a laboratory tool used to detect and quantify apoptosis, a process of programmed cell death. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, and 7-AAD, a DNA-binding dye, to identify cells undergoing early and late stages of apoptosis.
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76 protocols using annexin 5 apc 7 aad apoptosis detection kit
1
Apoptosis Detection by Flow Cytometry
2
Annexin V-APC/7AAD Apoptosis Assay
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HK-2 cells were detached by 0.25% Trypsin-EDTA digestion and collected by centrifugation at 1000 rpm for 5 min. The cells were re-suspended at the density of 1 × 106 cells/mL and stained with Annexin V-APC/7AAD Apoptosis Detection Kit (Keygentec, Nanjing, Jiangsu, China) according to the manufacturer’s instructions. Finally, the positive signals of apoptotic cells were analyzed with FACSCalibur flow cytometer (Becton Dickinson, San Diego, CA, USA).
3
Evaluating ESCC Cell Viability and Apoptosis
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Brie y, co-cultured PBMC and ESCC cells were digested into each well of a 96-well plate and incubated overnight in a nal volume of 200 µL. After that, cells were treated with 20µL of 5 mg/mL MTT (Invitrogen, USA) and incubated for 4 h at 37°C. Then, the MTT solution was removed, and added 200 µL of DMSO to each well. The absorbance was measured with a Spectrophotometer at 490 nm.
Apoptosis assay ESCC cells were co-cultured with PBMC. The cell apoptosis was assessed using the Annexin V-APC/7-AAD Apoptosis Detection kit (KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer's instructions. Brie y, add 500 µ L Binding Buffer, 5 µL Annexin V-APC, and 5 µL 7-AAD, followed by incubating at room temperature for 10 min in the darkness. Subsequently, the cells were analyzed by ow cytometry within 1h.
Apoptosis assay ESCC cells were co-cultured with PBMC. The cell apoptosis was assessed using the Annexin V-APC/7-AAD Apoptosis Detection kit (KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer's instructions. Brie y, add 500 µ L Binding Buffer, 5 µL Annexin V-APC, and 5 µL 7-AAD, followed by incubating at room temperature for 10 min in the darkness. Subsequently, the cells were analyzed by ow cytometry within 1h.
4
Cell Proliferation and Apoptosis Assays
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Cell proliferation was determined by using CCK-8 assay. SUDHL4 or OCI-LY7 cells were seeded at a density of 5 × 103 per well in 96-well plates and 10 µl of CCK-8 reagent was added and incubated at 37 ° C for 3 h. The absorbance (OD) at 450 nm was measured. Cell apoptosis was determined by annexin V-APC/7-AAD apoptosis detection kit (Keygen Biotech, China), Briefly, after transfection for 24 h, 48 and 72 h, cells were collected, stained with annexin V-APC and 7-AAD according to the manufacturer’s instructions, and then analyzed by flow cytometry. Each experiment was done in triplicate. For DNA histogram analysis, cells were stained with propidium iodide, and analyzed by flow cytometry.
5
Apoptosis Detection by Flow Cytometry
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Apoptosis was detected by flow cytometry using the Annexin V–APC/7-AAD Apoptosis Detection Kit (Keygen, China). Briefly, cells were harvested, washed, resuspended in the staining buffer, and doubly stained with annexin V and 7-amino-actinomycin D (7-AAD). For each experiment, 5 × 104 cells were analyzed using FACSCalibur and CellQuest software (BD Biosciences, USA). The annexin V–positive cells were regarded as apoptotic cells.
6
Cell Cycle and Apoptosis Analysis
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Treated cells were fixed in 75 % alcohol overnight at -20 °C for cell cycle assay. The cells were washed three times, and a Cell Cycle Analysis Kit (Beyotime, Shanghai, China) was used for propidium iodide (PI) staining.
For apoptosis analysis, all cells were treated with 0.5 mM H2O2 for 4 h to stimulate apoptosis. An Annexin VAPC/7-AAD Apoptosis Detection Kit (KeyGEN, Jiangsu, China) was used for Annexin V-APC and 7-AAD staining according to the manufacturer’s protocol. Finally, the cell cycle distribution percentage and the apoptotic rate were analyzed using BD FACSCanto II (BD Biosciences, San Jose, CA, USA).
For apoptosis analysis, all cells were treated with 0.5 mM H2O2 for 4 h to stimulate apoptosis. An Annexin VAPC/7-AAD Apoptosis Detection Kit (KeyGEN, Jiangsu, China) was used for Annexin V-APC and 7-AAD staining according to the manufacturer’s protocol. Finally, the cell cycle distribution percentage and the apoptotic rate were analyzed using BD FACSCanto II (BD Biosciences, San Jose, CA, USA).
7
Annexin V-APC/7-AAD Apoptosis Assay
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Annexin V-APC/7-AAD Apoptosis Detection Kit was obtained from Keygen Biotech (KGA1025) and Elabscience (E-CK-A218). Cell death was recorded on a FACSCalibur flow cytometer (Becton, Dickinson and Company) in the total population (10,000 cells), and the data were analyzed using FlowJo software (Version 7.6.1).
8
Quantification of Apoptosis by Flow Cytometry
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Apoptosis in cell cultures was quantitated by flow cytometry using Annexin V-APC/7-AAD Apoptosis Detection Kit (KeyGEN BioTECH (Jiangsu, China), KGA108). The fluorescence intensity of apoptotic cells was analyzed with a flow cytometer.
9
Evaluating Apoptosis via Annexin V-APC/7-AAD Assay
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Apoptosis was evaluated using the Annexin V-APC/7-AAD Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer's protocol. Briefly, cells (1×105/well) were seeded in a 96-well cell culture plate in RPMI-1640 medium with 10% FBS and incubated overnight at 37°C. They were then treated on the following day with 10 µM gefitinib or left untreated at 37°C in 5% CO2 and 95% humidified air for 48 h. Both adherent and suspended cells were harvested and washed twice with cold 1× phosphate-buffered saline (PBS), then resuspended in 500 µl binding buffer. Annexin V-APC (5 µl) and 7-AAD (5 µl) were added to 500 µl of the cell suspension, followed by incubation for 15 min in the dark. Samples were analyzed within 1 h on a Novocyte flow cytometer (ACEA Biosciences, San Diego, CA, USA). The experiment was performed using triplicate samples.
10
Apoptosis Evaluation of IMB-1406 in HepG2 Cells
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HepG2 cells were inoculated in 6-well plates and then processed accordingly after they adhered. Cells were treated with IMB-1406, after 72 h the cells were collected by trypsin without EDTA, washed twice with PBS. The Annexin V-APC/7-AAD Apoptosis Detection kit (KGA1024, KeyGEN, Nanjing, China) was used for cell staining and flow cytometry (Becton-Dickinson FACS Calibur) following the manufacturer's instructions. The percentage of apoptotic cells was determined using FACS flow cytometry and associated software (BECKMAN). Experiments were repeated three times, and the results are displayed as histograms.
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