APC annexin V / PI apoptosis Kit (C09-1097-451)

Annexin V is a Ca2+dependent phospholipid binding protein with a molecular weight of 35-36KD, which can selectively bind to phosphatidylserine (PS). Phosphatidylserine (PS) is mainly distributed on the inner side of the cell membrane, adjacent to the cytoplasm. In the early stages of cell apoptosis, different types of cells will flip phosphatidylserine onto the cell surface and expose it to the extracellular environment. At this point, using far-infrared fluorescent protein APC labeled Annexin V, i.e. Annexin V-APC, combined with everted phosphatidylserine (PS), can directly detect the everted phosphatidylserine, an important feature of cell apoptosis, using flow cytometry or fluorescence microscopy. For necrotic or late apoptotic cells, as cell integrity has been disrupted, Annexin V-APC can enter the cytoplasm and bind to PS located on the inner side of the phospholipid layer, resulting in far-infrared fluorescence of necrotic cells. Propidium Iodide (PI) is a DNA binding dye that can stain the nuclei of necrotic or late apoptotic cells that have lost membrane integrity. PI can be excited by laser at 488532 or 546 nm, exhibiting red fluorescence.Component：Usage:1. Experimental design:Blank tube: negative control group cells, without the addition of Annexin V-APC/PI. Used to regulate voltage.Single staining tube: Positive control group cells, with only Annexin V-APC/PI added. Used to adjust compensation.Detection tube: The processed cells were added with Annexin V-APC/PI. After adjusting the voltage compensation using blank tubes and single dye tubes, obtain the required flow data.2. Collect cells.(1) For suspended cells:a. After undergoing cell apoptosis stimulation, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect cells, gently resuspend cells with PBS and count them.Attention: PBS resuspension cannot be omitted. The process of PBS resuspension also plays a role in washing cells, ensuring the subsequent binding of Annexin V-APC.b. Take 5 × 104-1 × 105 resuspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 100 µ L 1 × Annexin V binding buffer to gently resuspend the cells.c. Add 5 µ L Annexin V-APC and gently mix well.d. Add 5 µ L of PI and gently mix well.e. Incubate at room temperature (20-25 º C) in the dark for 10-15 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 pancreatic enzyme cell digestion solution (without EDTA) to digest the cells. Incubate at room temperature until gently blowing can remove the digestive fluid of pancreatic enzyme cells when the adherent cells are blown down. It is necessary to avoid excessive digestion of pancreatic enzymes.Note: For adherent cells, the pancreatic enzyme digestion step is crucial. If the digestion time of pancreatic enzymes is too short, the cells need to be vigorously blown to fall off, which can easily cause damage to the cell membrane and lead to false positive cell necrosis; If the digestion time is too long, it is also easy to cause cell membrane damage and false positive cell necrosis, and even affect the binding of phosphatidylserine and Annexin V-APC 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 with PBS and count.Note: Adding the cell culture medium mentioned in the previous step is very important. On the one hand, it can collect cells that have undergone apoptosis or necrosis that have already been suspended. On the other hand, the serum in the cell culture medium can effectively inhibit or neutralize residual pancreatic enzymes. The residual pancreatic enzyme will digest and degrade the subsequent addition of Annexin V-APC, 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 1 × Annexin V binding buffer to gently resuspend the cells.d. Add 5 µ L Annexin V-APC and gently mix well.e. Add 5 µ L of PI and gently mix well.f. Incubate at room temperature (20-25 º C) in the dark for 10-15 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 can be directly added μ Resuspension cells with L 1 × Annexin V binding buffer and immediately detect on the machine. Annexin V-APC is excited by 633 nm, and the fluorescence emission spectrum is detected at 660 nm (FL4 or RL1 channel). PI is excited at 488 nm and the detected emission spectrum is approximately at 617 nm (FL3 or BL3 channel).b. On the scatter plot of the bivariate flow cytometer, the lower left quadrant displays live cells as (Annexin V-APC -, PI -); The lower right quadrant represents early apoptotic cells, which are (Annexin V - APC+/PI -); The upper right quadrant shows necrotic and late apoptotic cells, which are (Annexin V-APC+, PI+); The upper left quadrant displays naked nuclear cells, which are (Annexin V-APC -, PI+).(2) Fluorescence microscopy detection:a. Centrifuge at 1000 rpm for 5 minutes, collect cells, and gently resuspend them with 400 µ L 1 x Annexin V binding buffer. After settling the cells in a 96 well plate or performing cell smear, observe them under a fluorescence microscope.b. Annexin V-APC can use filters suitable for APC, while PI can use filters suitable for Cy3 or Texas.Product parameters:Annexin v-apc: ex/em = 650/660 nmpi: ex/em=535 nm/617 nm (with DNA)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 intake of rednucleus II; 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.Scope of application:Early apoptosis detection, annexin V Kit.