| Operating Instructions |
(1) Centrifuge at low speed before use to avoid liquid accumulation in the tube cover and tube wall;
(2) This reagent is only suitable for experiments, not for clinical testing;
(3) PI (propidium iodide, propidium bromide) is toxic, can be absorbed through the skin, and has an irritating effect on the eyes. Wear gloves when using it;
(4) Annexin V-FITC and PI are photosensitive substances, please pay attention to avoid light during operation. When handling and marking, do it in the dark as much as possible. During the incubation phase, the container is wrapped with aluminum foil or placed in a drawer. After the cells were labeled, they were observed with a microscope in a dark room;
(5) The whole operation process should be as gentle as possible, do not blow the cells hard, and try to operate at 4 ℃ to avoid affecting the cell state.
(6) In the last step of cell washing, please discard the supernatant as much as possible to avoid PBS residue, which may affect the experimental results.
(7) In order to prevent fluorescence decay, flow cytometry detection should be performed within 1 hour.
(8) If the PI staining time is too long, it may cause the detected apoptosis rate to be high. It is recommended to perform Annexin V-FITC staining first, and PI staining can be added at least 5 minutes before the machine.
|
| Beilstein |
0 |
| Synonym |
Annexin V-FITC apoptosis assay kit |
| Protocol |
Adherent cells need to be digested with 0.25% trypsin. Note that over-digestion can damage cells. 2% BSA can be added during digestion to prevent overdigestion. If digested with trypsin containing EDTA, be careful that EDTA must be completely removed: wash with 1 × PBS or 1 × binding buffer before labeling to remove EDTA to prevent residual EDTA from chelating with Ca2 + and affecting the binding of Annexin V.
(1) Dilute 10 × Binding Buffer into 1 × Binding Buffer with deionized water;
(2) Cell collection: suspension cell collection: centrifugation for 5 minutes; Adherent cells: After digestion and collection with trypsin without EDTA (Note: the trypsin digestion time should not be too long, otherwise it will affect the binding of phosphatidylserine and Annexin V-FITC on the cell membrane), centrifuge at 2000 rpm at room temperature for 5 ~ 10 minutes to collect the cells;
(3) Cell washing: Resuspend the cells once with pre-cooled 1 × PBS (4 ℃), centrifuge at 2000 rpm for 5-10 minutes, and discard the supernatant to wash the cells;
(4) 300 μL of 1 × Binding Buffer suspension cells were added;
(5) Annexin V-FITC labeling: Add 5μL of Annexin V-FITC and mix well, protect from light, and incubate at room temperature for 15 minutes;
(6) PI labeling: Add 5μL of PI staining 5 minutes before loading the machine.
(7) Before loading, add 200 μL of 1 × Binding Buffer.
Note: The recommended number of cells per reaction system is 1 × 10
4To 1 × 1061 |
| Description |
In normal cells, phosphatidylserine is only distributed on the inside of the lipid bilayer of the cell membrane. At the earliest stage of cell apoptosis, membrane phosphatidylserine (PS) turns from the inside to the outside of the lipid membrane. This change is earlier than apoptotic phenomena such as cell shrinkage, chromatin condensation, DNA fragmentation and increased permeability of the cell membrane. Annexin V is a phospholipid-binding protein with 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. Propidium Iodide (PI) is a nucleic acid dye, which cannot penetrate the intact cell membrane. However, due to the increase in cell membrane permeability of cells in the middle and late stages of apoptosis and dead cells, PI can penetrate the cell membrane and make the cell nucleus red. Therefore, matching Annexin V with PI can distinguish cells in different apoptotic stages. |
| Component |
|
25 Tests |
50 Tests |
100 Tests |
| AnnexinV-FITC |
125μl |
250μl |
500μl |
| 10×Binding Buffer |
1.5ml |
2.5ml |
6.5ml |
| Propidium Iodide |
125μl |
250μl |
500μl |
|
| PubChem CID |
0 |
| Storage Temp. |
4 °C for 6 months; Avoid repeated freezing and thawing. |
