Apoptosis detection kit-TUNEL detection method

In fact, in a strict lexical sense, there is a big difference between programmed cell death (PCD) and apoptosis. Programmed cell death is a functional concept that describes certain cell deaths in a multicellular organism as a predetermined and strictly controlled normal part of ontogeny. Apoptosis is a morphological concept, which describes a form of cell death that is completely different from necrosis with a complete set of morphological characteristics. Morphological changes of cell apoptosis: cell membranes and organelles are relatively complete. Nucleus pyknosis from left to right is nuclear shrinkage, nuclear lysis, and nuclear fragmentation. Next, let’s learn about the detection-measurement-method-method of apoptosis: According to classification, the detection methods of apoptosis include morphological detection, cell function detection, and biochemical marker detection. So what are the advantages and disadvantages of these methods? 1. Morphological detection methods: 1) Observation of the submicroscopic structure of organelles, electron microscope: Advantages: The gold standard for apoptosis detection. Disadvantages: unable to quantify; cumbersome experimental procedures 2) Nucleus staining and microscopic examination (Hoechst 33342) Advantages: low cost, convenient operation, and quantifiable. Disadvantage: Need to count the cells. 2. Cell function detection method: 1) Mitochondrial membrane potential detection JC-1, MitoTracker advantages: suitable for imaging detection, staining results can only be used to determine the line. Disadvantages: Whether the mitochondrial membrane potential is lost, it cannot be quantified. 2) Detection of phosphatidylserine on cell membrane surface. Advantages of Annexin V-AbFluor 488/PI: a popular method for detection of apoptosis. Disadvantages: It is necessary to prepare a single cell suspension to quantify the proportion of apoptosis in different periods. 3. Biochemical marker detection method 1) Apoptosis molecular marker detection Caspase activity detection Caspase content detection (WB) advantages: high-throughput screening and quantitative. 2) DNA damage detection. Advantages of DNA electrophoresis TUNEL method: It is suitable for the apoptosis study of tissue section samples. Disadvantages: electrophoresis detection is time-consuming, unable to quantify, and poor stability. Combination detection method for accurate apoptosis detection 1. The Annexin V-AbFluor 488/PI detection method uses Annexin V-AbFluor 488 (green fluorescence) and PI (red fluorescence) together, which can accurately distinguish early apoptosis and apoptosis Late and dead cells. For this method, sample processing, staining operations, and fluorescence photography determine the accuracy of apoptosis detection. For samples that are not used and different detection methods, it is recommended to follow the following valuable experience: 1. The key to fluorescence photography 1) Suspension or resuspension Take pictures of liquid slides. Resuspend the cells with a small amount of PBS or resuspension solution to form a cell concentrate. Pipette 5-10ul on a clean anti-removal glass slide to make a circle smear. Cover the cover slip and immediately under the microscope. Perform a microscopic examination. Pay attention to the speed during the processing to prevent the cells from dying. The resuspension density is not too small. When smearing, the cell suspension volume should be less than 10ul. The entire operation is recommended to be completed within 10 minutes. 2) There is a small amount of buffer at the bottom of the cell culture small dish or the photo-climbing dish to avoid dry slices. Be sure to clean gently before taking pictures to prevent inadequate cleaning from causing non-specific background during the photo process. Hela cells were induced with camptothecin for 24 hours, and stained with Annexin V-AbFluor 488 Apoptosis Detection Kit (KTA0002). Can be stained by Annexin V-AbFluor 488/PI (green membrane and red fragment nucleus) are early apoptotic cells, necrosis or late apoptotic cells. 2. Key steps of flow cytometry 1) Avoid false positives when digesting cells. To prevent false positives, trypsin without EDTA is recommended for digesting cells. 2) Set the control group. The amount measured in flow cytometry is a relative value, not a value. If you want to know the value, you must set up a control group, which includes a negative control and a positive control. It is recommended to set a blank group and Annexin V-AbFluor 488/PI single staining group control group when testing on the machine, and the flow cytometer needs to be adjusted in advance. 3) The amount of cells to be tested should be moderate. It is recommended to prepare a sufficient amount of cells. Generally, it is recommended to prepare 1×106 cells. If there are too few cells, the sample flow will increase and the coefficient of variation will be affected. The result is not accurate. Relatively insufficient antibodies or dyes, the results are also affected. Camptothecin induces Hela cells for 24 hours and detects them with Annexin V-AbFluor 488 Apoptosis Detection Kit (KTA0002, Abbkine). Using Annexin V-AbFluor 488 and propidium iodide PI can distinguish early apoptotic cells (Annexin V-AbFluor 488 positive), late apoptotic or necrotic cells (Annexin V-AbFluor 488 and propidium iodide positive). 2. TUNEL detection method 1. Key steps in the tissue TUNEL experiment 1) Fully dewaxing and hydration <TIPS> Before dewaxing, the slices can be baked in an oven at 65°C for 30 minutes, and then dewaxed with xylene for 10-20 minutes. For hydration, gradient ethanol was used to immerse from high concentration to low concentration for 5 minutes. 2) Grasp the time for cell permeation <TIPS> Choose the incubation time of proteinase K according to the thickness of the slices. The concentration of proteinase K should not be less than 20 μg/mL. It is usually treated at 37°C for 10-30 min, and the 6um slice time is slightly shorter. The 20um slice increases the time, through exploration, it is possible to not only not fall off the slice, but also enable the subsequent enzymes and antibodies to enter the cell. If the treatment time is too long, the film will easily fall off, and if the treatment time is too short, the transparent effect cannot be achieved. 3) Properly extend the time of the TUNEL reaction solution <TIPS> The recommended treatment condition is 37°C for 2 hours. A longer time can be selected according to the estimated degree of apoptotic damage. It is recommended to use the final background coloring for judgment. 4) Full cleaning of PBS <TIPS> The cleaning after the TUNEL reaction should be very strict. It is recommended to use PBST to repeat the cleaning and then change to PBS for thorough cleaning, or increase the number of PBS washes, because these cleanings directly determine the non-specific staining of the last slice. Paraffin section of mouse intestinal tissue, 200 times magnification, red fluorescence is the cell that undergoes late apoptosis. Reagents used: KTA2011 TUNEL method apoptosis detection kit (orange fluorescence), DAPI operation flow chart: 2. Key steps in the cell TUNEL experiment 1) Judgment and processing of the dosing model <TIPS> Cell samples must be confirmed when TUNEL is done Whether the model is successful or not, it is necessary to be able to clearly judge the apoptotic cells during white light microscopy. 2) The use of proteinase K <TIPS>The purpose of proteinase K is to permeate the cell membrane and nuclear membrane, so that the reagents can fully enter the nucleus for reaction and increase the positive rate. If the concentration is too high or the incubation time is too long, it is easy to cause the cells to fall off. It is recommended to use 20 μg/mL proteinase K (0.5% Triton X-100), and the reaction time is recommended to be 5-15 min. Note: The key reagent proteinase K must find the most suitable conditions for use. If the temperature is too high, the reaction time is too long, it is easy to damage the nucleic acid structure, and false positives will occur.