Annexin V-APC/PI apoptosis analysis kit
Annexin V-APC/PI apoptosis analysis kit
Product Details
Product Details
Product Specification
| Usage |
1. Self-prepared instruments and reagents: 1. Reagent: PBS (abs962); EDTA-free trypsin digestion (abs47014937, abs47048223) 2. Consumables and instruments: 1.5 mL centrifuge tube (sterile and enzyme-free) (abs7119), flow cytometer, fluorescence microscope, low-speed centrifuge, micropipette. 2. Operation steps: For streaming operation detection 1. Cell collection (1) Suspended cells: 300 × g centrifugal force, centrifugation for 5 min to collect; (2) Adherent cells: Adherent cells are collected by trypsin digestion without EDTA (Note: the trypsin digestion time should not be too long, otherwise it will easily cause false positives); 2. Wash the cells twice with PBS (300 × g, centrifuge for 5 min) and collect 1-5 × 105Cells; 3. Add 500μL of Binding Buffer and gently blow to form a single cell suspension; 4. Add 5μL Annexin V-APC and 5μL Propidium Iodide, and gently mix; 5. React for 5-10 min at room temperature and in the dark from light; 6. Please perform flow cytometer observation and detection within 1 hour. 7. Flow cytometry analysis (1) Detection by flow cytometry, fluorescence channel (FL4) of Annexin V-APC (Ex = 633 nm, Em = 660 nm); PI red fluorescence (Ex = 488 nm, Em ≥ 630 nm) was detected by PE channels (FL2 or FL3). (2) Fluorescence compensation adjustment: Using induction-treated apoptotic cells as a control, fluorescence compensation adjustment was performed to remove spectral overlap and set the position of cross gates. For fluorescence microscopy detection 1. Select a 24-well plate or a 48-well plate for cell culture and treatment; 2. Before staining, use a multi-well plate centrifuge 300 × g for 5 min; 3. Remove the cell culture medium and add PBS to wash once; 4. According to the number of samples, calculate and prepare the staining working solution: in a 1.5 mL EP tube, add 500μL Binding Buffer, 5μL Annexin V-APC and 5μL Propidium Iodide, and mix well (take 500μL as an example); 5. Add the staining working solution to the well plate (500μL per well for the 12-well plate, 200μL per well for the 24-well plate, 100μL per well for the 96-well plate), and incubate at room temperature in the dark for 20 minutes; 6. Observe immediately under a fluorescence microscope. |
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| Beilstein | 0 | ||||||||||||||||||||
| Synonym | Annexin V-APC/PI Apoptosis Analysis Kit | ||||||||||||||||||||
| Description |
In normal cells, phosphatidylserine (PS) is only distributed on the inside of the lipid bilayer of the cell membrane, while in the early stage of apoptosis, phosphatidylserine (PS) in the cell membrane turns from the inside to the outside of the lipid membrane. Annexin V is a Ca2 +-dependent phospholipid binding protein with a molecular weight of 35-36 kD. It has a high affinity for phosphatidylserine, so it can bind to the cell membrane of early apoptotic cells through the exposed phosphatidylserine on the outside of the cell. Therefore, Annexin V is one of the sensitive indicators to detect early apoptosis. Annexin V was labeled with fluorescein APC, and the labeled Annexin V was used as a fluorescent probe to detect the occurrence of apoptosis by fluorescence microscope or flow cytometry.
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| PubChem CID | 0 | ||||||||||||||||||||
| General Notes | 1. Please centrifuge and collect the solution before taking the trace reagent. 2. Annexin V-APC, Propidium Iodide (PI) is stored and used in the dark from light. 3. Propidium Iodide (PI) is toxic, please wear gloves when operating. 4. This kit is suitable for detecting living cells. When detected by flow cytometry, the number of cells should not be less than 1 × 105Units/mL, not recommended for testing tissue samples. 5. Adherent cells need to be digested with trypsin without EDTA. If digested improperly, it may cause false positives, and using a cell scraper will cause cells to adhere and form clusters, which will affect the detection. Trypsinized cells can be stored in PBS containing 2% (w/v) BSA to prevent further damage. 6. The fluorescence may be quenched after cell fixation. Please do not fix the sample. 7. Due to different types of detected cells, types of apoptosis inducers, detection instruments used, and different setting parameters, it is recommended that cells treated with apoptosis induction should be used as controls for each detection to adjust fluorescence compensation. |
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| Storage Temp. | Keep as concentrated solution. Store at 4°C and protected from prolonged exposure to light. Do not freeze.The validity period is 6 months. |
