| Product Description |
Annexin V (annexin-V) is a ca2+ - dependent phospholipid binding protein with a molecular weight of 35-36 KD, which can selectively bind to phosphatidylserine (PS). Phosphatidylserine (PS) is mainly distributed on the inner side of the cell membrane, that is, the side adjacent to the cytoplasm. At the early stage of apoptosis, different types of cells will evert phosphatidylserine to the cell surface and expose it to the extracellular environment. At this time, annexin v-pe labeled with fluorescent protein PE is combined with everted phosphatidylserine (PS), and the everted phosphatidylserine, an important feature of apoptosis, can be directly detected by flow cytometry. Normal cells will not be stained by annexin v-pe, and cells undergoing apoptosis or necrosis will be stained by annexin v-pe. Annexin v-pe can be combined with a partially impermeable nuclear dye (7-aad/pi) to distinguish cells at different stages of apoptosis. Product parameters： Annexin v-pe:ex/em = 488 / 578 nm Matters needing attention： 1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. to reduce the process of apoptosis, the incubation process can be operated on ice, but the incubation time should be extended to at least 30 min. 3. as apoptosis is a rapid process, it is recommended that samples be analyzed within 1 h after staining. 4. for adherent cells, digestion is a key step. If there are floating cells when adherent cells induce apoptosis, the floating cells and adherent cells should be collected and stained. Handle adherent cells with care to avoid artificial damage to cells. The trypsin digestion time is too short, and the cells need to be blown hard to fall off, which is easy to cause damage to the cell membrane and excessive PI intake; If the digestion time is too long, the cell membrane is also prone to damage, and even affect the binding of phosphatidylserine and annexin v-pe on the cell membrane. When digesting, spread pancreatin on the bottom of the well plate, fully contact the pancreatin with the cells when shaking gently, then pour out most of the pancreatin, use the remaining small amount of pancreatin to digest for a period of time, and terminate when the gap between cells increases and the bottom of the bottle is spotted. Try not to use EDTA in the digestive juice, which will affect the binding of annexin V to PS. 5. after the adherent cells are digested with trypsin, it is recommended to stain after recovering in the optimal culture conditions and medium for about 30 min to avoid false positives. 6. in order to avoid losing cells when washing cells, you can use a large tip over a small tip to aspirate. 7. the optimal concentration of dye is determined by the specific experimental requirements. 8. fluorescent dyes have quenching problems. Please try to avoid light during storage and use to slow down fluorescence quenching. 9. for your safety and health, please wear experimental clothes and disposable gloves. Usage method:
1. Induce cell apoptosis according to experimental requirements. The test sample should include untreated cell samples as negative controls. In addition, set a set of samples for single dyeing to adjust compensation.
2. Collect cells
(1) For suspended cells:
a. After inducing cell apoptosis, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS, and count them.
Note: PBS resuspension cannot be omitted. The process of PBS resuspension also serves to wash cells, ensuring the subsequent binding of Annexin V-PE.
b. Take 5 × 104-1 × 105 resuspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 100 µ L of 1 × Annexin V binding buffer (optional final concentration of 2 mM Hepes/NaOH, pH 7.4, 28 mM NaCl, 0.5 mM CaCl2 solution) to gently resuspend the cells.
c. Add 5 µ L Annexin V-PE and mix gently.
d. Add 10 μ L of 20 μ g/mL 7-AAD or PI and mix gently.
e. Incubate at room temperature (20-25 º C) in the dark for 15 minutes. Aluminum foil can be used to avoid light. During the incubation process, cells can be resuspended 2-3 times to improve staining efficiency.
(2) For adherent cells: a. Suck out the cell culture medium into a suitable centrifuge tube, wash the adherent cells with PBS once, and add an appropriate amount of trypsin cell digestion solution (without EDTA) to digest the cells. Incubate at room temperature until gently blowing can remove the trypsin cell digestion solution when the adherent cells are blown down. Overdigestion of pancreatic enzymes should be avoided.
Note: For adherent cells, the trypsin digestion step is crucial. If the trypsin digestion time is too short, cells need to be blown hard to detach, which can easily cause damage to the cell membrane and lead to false positives of cell necrosis; If the digestion time is too long, it can also cause cell membrane damage and false positives of cell necrosis, and even affect the binding of phosphatidylserine and Annexin V-PE on the cell membrane, thereby interfering with the detection of cell apoptosis.
b. Add the cell culture medium collected in the previous step, gently blow down the cells, transfer them to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS and count them.
Note: Adding the cell culture medium from the previous step is very important. On the one hand, it can collect cells that have already been suspended and undergone apoptosis or necrosis. On the other hand, the serum in the cell culture medium can effectively inhibit or neutralize residual trypsin. The residual trypsin will digest and degrade the subsequently added Annexin V-PE, leading to staining failure.
c. Take 5 × 104-1 × 105 resuspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 100 µ L of 1 × Annexin V binding buffer (optional final concentration of 2 mM Hepes/NaOH, pH 7.4, 28 mM NaCl, 0.5 mM CaCl2 solution) to gently resuspend the cells.
d. Add 5 µ L Annexin V-PE and mix gently.
e. Add 10 μ L of 20 μ g/mL 7-AAD or PI and mix gently.
f. Incubate at room temperature (20-25 º C) in the dark for 15 minutes. Aluminum foil can be used to avoid light. During the incubation process, cells can be resuspended 2-3 times to improve staining efficiency. 3. Result analysis:
(1) Flow cytometry detection:
a. After incubation, 400 μ L of PBS can be directly added to resuspend the cells and immediately detected on the machine. When detected by flow cytometry, Annexin V-PE was excited by 488 nm/566 nm laser, and the fluorescence emission spectrum was detected at 578 nm (BL2 (FL2)/YL1 channel).
b. On the scatter plot of the bivariate flow cytometer, live cells are shown in the lower left quadrant as (Annexin V-PE -, 7-AAD/PI -); The lower right quadrant represents early apoptotic cells, which are (Annexin V-PE+, 7-AAD/PI -); The upper right quadrant represents necrotic and late apoptotic cells, which are (Annexin V-PE+, 7-AAD/PI+); The upper left quadrant displays naked nuclear cells, which are (Annexin V-PE -, 7-AAD/PI+).
(2) Fluorescence microscopy detection:
a. Centrifuge at 1000 rpm for 5 minutes, collect cells, and gently resuspend them in 400 µ L of 1 × Annexin V binding buffer. Transfer the cells to a 96 well plate and settle for a moment or perform cell smear, then observe under a fluorescence microscope.
b. Annexin V-PE can use filters suitable for PE.
Scope of application： Early apoptosis detection, annexin v-coupled fluorescent protein |
K2412248