Annexin V-FITC/PI Apoptosis Detection Kit (C09-0728-612)

Product descriptionPhosphatidylserine (PS) is a negatively charged phospholipid. In normal cells, PS is only distributed inside the lipid bilayer of the cell membrane. In the early stage of apoptosis, the cell membrane PS turns from the inside of the lipid membrane to the outside of the cell membrane. PS is exposed on the outer surface of the cell membrane. Annexin V is a Ca2+-dependent phospholipid binding protein with a molecular weight of 35~36kD. It has a high affinity for phosphatidylserine, so it can bind to the cell membrane of early apoptosis cells through phosphatidylserine exposed outside the cell. Therefore, Annexin V is recognized as one of the sensitive indicators for detecting early cell apoptosis.The Annexin V is labeled with green fluorescence (FITC), and the labeled Annexin V is used as a probe to detect the occurrence of cell apoptosis using a fluorescence microscope or flow cytometer. Propidium Iodide (PI) is a nucleic acid dye. It cannot penetrate the complete cell membrane of normal cells or early apoptotic cells. However, PI can penetrate the cell membrane in the middle and late stages of apoptosis and necrotic cells. Stain the nucleus red. Therefore, the method of double staining with Annexin V and PI can distinguish cells in different stages of apoptosis.                     Figure 1. Schematic diagram of phosphatidylserine (PS) eversion during cell apoptosisKit componentsReagents20 assays50 assays100 assaysStorageAnnexin V-FITC100 μl250 μl500 μl4°C Protect from lightPropidium Iodide, PI200 μl500 μl1000 μl4°C Protect from lightBinding Buffer ( 4X )4 ml10 ml20 ml4° Product parametersEx(nm): 490    Em(nm): 520Required laboratory equipmentMicropipette; PBS; trypsin digestion solution without EDTA; low-speed centrifuge; flow cytometerPrecautions1. This kit is for research use only.2. Micro-reagents need to be centrifuged for a few seconds to collect the reagents to the bottom of the tube before opening the cap for use.3. Propidium Iodide (PI) is toxic. Wear gloves when handling it and avoid contact with skin, eyes and mucous membranes.4. Annexin V-FITC contains sodium azide (NaN3), a highly toxic ingredient. Wear gloves when handling and avoid contact with skin, eyes and mucous membranes.5. This kit is used to detect live cells, and the number of cells should not be less than  1x10^6 during flow cytometry.6. It is advisable to test as soon as possible after staining, too long time may lead to an increase in the number of apoptosis or necrotic cells.7. The Annixin V method for detecting apoptotic cells is suitable for the detection of cells growing in suspension, such as lymphocytes. For adherent cells, the cell membrane will be damaged during the digestion process such as trypsin, which will cause high false positives. The use of cell scrapers will cause the cells to adhere and form clusters, which will affect the detection. The digested cells are stored in PBS containing 2% BSA to prevent further damage. Although at present, some units including foreign countries also use this method to detect adherent growth of cells. I do not recommend using this method to detect. Because of its poor repeatability and the need to be very careful when operating.8. Trypsin remaining after digesting adherent cells will digest and degrade Annexin V-FITC, which will eventually lead to staining failure.9. After the cells are fixed, fluorescence may be quenched. Please do not fix the sample.Instructions:1. Preparation of cell samples:a) Suspension cells:1) Collect the cells into a centrifuge tube and centrifuge at 1000-2000rpm for 5min, carefully remove the supernatant.2) Gently resuspend the cells with 1ml of 4°C pre-cooled PBS and count, centrifuge at 1000-2000rpm for 5min, and remove the supernatant by suction.3) Add 1ml of 4°C pre-cooled PBS to resuspend the cells, centrifuge at 1000-2000rpm for 5min, and remove the supernatant by suction.b) Adherent cells:1) Aspirate the cell culture medium into a centrifuge tube, wash the adherent cells once with PBS, and add an appropriate amount of EDTA-free trypsin cell digestion solution to digest the cells.2) Incubate at room temperature until the adherent cells can be removed by gentle pipetting, and then aspirate the trypsinized cell digestion solution. Over-digestion with trypsin should be avoided.3) Add the cell culture medium collected in the above steps, mix it slightly, transfer it to a centrifuge tube, centrifuge at 1000-2000rpm for 5min, and remove the supernatant by suction.Note: On the one hand, the added cell culture medium can collect suspended apoptotic or necrotic cells, and on the other hand, the serum in the cell culture medium can effectively inhibit or neutralize the residual trypsin; Enzymes will digest and degrade subsequently added Annexin V-FITC causing staining to fail.4) Gently resuspend the cells with 1ml of 4°C pre-cooled PBS and count, centrifuge at 1000-2000rpm for 5min, and remove the supernatant by suction.5) Add 1ml of 4°C pre-cooled PBS to resuspend the cells, centrifuge at 1000-2000rpm for 5min, and remove the supernatant by suction.2. Dilute the Binding Buffer 1:4 with deionized water (4ml Binding Buffer+12ml deionized water);3. Resuspend the cells with 250µl of binding buffer to adjust the concentration to 1×106/ml;4. Take 100µl of cell suspension into a 5 ml flow tube, add 5µl Annexin V-FITC, and mix gently;5. Incubate at room temperature (20-25°C) for 10 minutes in the dark;6. Add 10µl propidium iodide solution 5 minutes before putting on the machine, and mix gently;7. Add 400µl PBS to the reaction tube to resuspend the cells before putting on the machine, store in the dark, and then perform flow cytometry (FACS) detection, Annexin V-FITC is green fluorescence, PI isRed fluorescence. Related Document: https://ald-pub-files.oss-cn-shanghai.aliyuncs.com/aladdinsci/pdp/sds/1/A266226-SCI_60615e75c846dffeb61b8c18f9046590.pdf