SACC‐LM or SACC‐83 were seeded in 6‐well plate with 2 × 105 cells per well. The apoptosis was assessed with Annexin V‐PE apoptosis detection kit following its protocol (KeyGEN BioTECH). The cells were harvested and stained by Annexin V‐PE or/and PI for 15 min at room temperature and were analysed with FACSCalibur flow cytometer.
Annexin 5 pe apoptosis detection kit
Manufactured by Keygen Biotech
Sourced in China
The Annexin V-PE Apoptosis Detection Kit is a laboratory tool designed to detect and quantify apoptosis, a programmed cell death process. The kit utilizes Annexin V, a calcium-dependent phospholipid-binding protein, conjugated with the fluorescent dye Phycoerythrin (PE) to identify cells undergoing apoptosis. This product provides a straightforward method to measure apoptosis in cell samples.
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8 protocols using annexin 5 pe apoptosis detection kit
1
Annexin V-PE Apoptosis Assay Protocol
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Annexin V-PE and 7-AAD Apoptosis Assay
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Cells were spun down to remove supernatant and resuspended in 100 μl Annexin V binding buffer. Then 5 μl of Annexin V-PE and 5 μl 7-AAD from the Annexin V-PE Apoptosis Detection Kit (Keygen Biotech) were added into the solution according to the guidelines. After 15 min of incubation, 400 μl of Annexin V binding buffer was added. The Annexin V-PE and 7-AAD stained cells were analyzed by the FL2 and FL4 channels.
3
Annexin V-PI Apoptosis Assay in HBE Cells
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HBE cells were seeded in 6-well plates at a density of 3×105 cells/well. Cells treated with drugs or the blank diluent were rinsed with warm PBS (37°C). The cell suspensions were washed twice with ice-cold PBS before further processing. The cells were stained using an Annexin V/PI staining kit (KeyGEN Annexin V-PE Apoptosis Detection Kit); 500 μl of binding buffer was added to each tube and transferred to a 1.5 ml centrifuge tube (1–5×105 cells). Then, 5 μl of Annexin V–FITC and 5 μl of PI were added, and the cells were gently vortexed. Cells were then incubated for 15 min at room temperature (RT) in the dark. Finally, the cells were analyzed by flow cytometer after 1 h (Becton Dickinson) (Ex 488 nm, and Em 530 nm).
4
Annexin V-PE Apoptosis Assay in HeLa Cells
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The HeLa cells were transfected with scramble siRNA and GAPDH siRNA for 48 h, treated with ETO for 4 h, and then collected and stained with both Annexin V and PE using an Annexin V-PE Apoptosis Detection Kit (KeyGEN BioTECH, Cat#KGA1011, Nanjing, China) according to the manufacturer’s instructions. The apoptosis was analyzed by flow cytometry using the BD FACSverse.
5
Cell Proliferation and Apoptosis Assays
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According to the manufacturer’s instructions, CCK8 (Beyotime, China) was used to measure the proliferation of H460-vshUSP7, A549-vshUSP7, and control cells. The specific process was previously described [9 (link)].
For the colony formation assay, cells were planted into three 6-cm cell culture dishes (1,000 cells per dish) and incubated for 12 days. Plates were washed with PBS then stained with Giemsa. Colonies with more than 50 cells were counted.
According to the manufacturer’s instructions, an Annexin V-PE Apoptosis Detection Kit (KeyGEN Biotech, China) was used to measure apoptosis in the H460, H460-vshUSP7, A549, and A549-vshUSP7 cells. First, 2 × 105 cells were collected and then washed twice with PBS. Cells were then resuspended in 500 μl binding buffer and transferred to the EP tube. After adding 1 μl Annexin V-PE and mixing at room temperature, samples were placed in the dark for 15 min. Cell apoptosis was then detected by flow cytometry. Experiments were repeated in triplicate.
For the colony formation assay, cells were planted into three 6-cm cell culture dishes (1,000 cells per dish) and incubated for 12 days. Plates were washed with PBS then stained with Giemsa. Colonies with more than 50 cells were counted.
According to the manufacturer’s instructions, an Annexin V-PE Apoptosis Detection Kit (KeyGEN Biotech, China) was used to measure apoptosis in the H460, H460-vshUSP7, A549, and A549-vshUSP7 cells. First, 2 × 105 cells were collected and then washed twice with PBS. Cells were then resuspended in 500 μl binding buffer and transferred to the EP tube. After adding 1 μl Annexin V-PE and mixing at room temperature, samples were placed in the dark for 15 min. Cell apoptosis was then detected by flow cytometry. Experiments were repeated in triplicate.
6
Apoptosis Evaluation in Myeloma Cells
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After treatment with platelet-rich plasma or exosomes for 48 h, the RPMI8226 and U266 cells were obtained by centrifugation at 2000 × g for 5 min. A Annexin V-PE apoptosis detection kit (KeyGen Bio Tech, Nanjing, China) was used to measure the apoptotic cells according to the instructions. The results were analyzed by using BD FlowJo software (V10). The apoptotic cells included annexin V+ single positive cells plus annexin V+ PI+ positive cells.
7
Annexin V-PI Apoptosis Assay
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SMMC-7721 cells were harvested by trypsinization and underwent centrifugation at 1000 g for 5 min. After washing twice with cold PBS, the cells were incubated with 500 μl 1 × binding buffer containing 5 μL Annexin V and 5 μL PI (KeyGEN Annexin V-PE Apoptosis Detection Kit) in the dark for 15 min. Finally, apoptosis was analyzed by flow cytometer (FACScan, Becton Dickinson, San Francisco, CA, USA).
8
Annexin V-PE Apoptosis Assay
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The percentage of apoptotic cells was determined by using an Annexin V-PE apoptosis detection kit (KeyGen Biotech, Jiangsu, China) according to the manufacture’s introduction. In brief, a total of 105 T24 and BIU cells were harvested after incubation with FL3 for 24 h, then stained these cells with Annexin V and PE subsequently. Finally, the cells were subjected to apoptosis analysis with the ACEA NovoExpress System (ACEA Biosciences Inc., CA, USA).
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