Hoechst staining method: Hoechst can pass through the living cell membrane and bind to the nucleus. There are two types of hoechest33342 and hoechst33258 (mainly apoptotic living cells) to stain the nucleus blue under ultraviolet light. Hoechst staining of the nucleus will affect the confocal microscope's observation of other fluorescence in this sample. Hoechsthoechsts33258 and hoechst33342 are not much different, but hoechst33342 has less toxic effect on cells, so in general hoechsts33258 is used for staining after cell fixation, while hoechst33342 can directly stain live cells! Staining step PI (PropidiumIodide Propidium Iodide) staining: is a kind of nuclear staining (PropidiumIodide, PI) that can stain DNA. It is a nucleic acid dye (red reagent, commonly used for cell apoptosis detection. Propidium iodide color), It cannot penetrate the complete cell membrane, but due to the increased permeability of the cells in the middle and late stages of apoptosis and necrotic cells, PI can penetrate the cell membrane and stain the cell nucleus red. Observing the cultured cells with PI single staining can only indicate cell necrosis, not apoptosis (of course, late apoptosis PI can also be stained). But if you just want to know the death of the cells, rather than carefully distinguish between necrosis or apoptosis, then PI single staining is also possible. But if you must determine the apoptosis of cells, then PI single staining is obviously not enough! The annexin-v staining cell apoptosis early, cell membrane markers changed. Among them, phosphatidylserine (Annexin-V, PS) is everted, and Annexin-V specifically binds to its high affinity in the presence of Ca2 +. In this way, Annexin-v staining is positive, indicating that the cells are in an early stage of apoptosis. Annexin-V combined with different fluorescent antibodies can use flow cytometry, fluorescence microscopy and confocal laser scanning microscopy to detect the occurrence of apoptosis. AnnexinV fluoresces green with FITC label; red if it is labeled with PE. JC-1 staining JC-1 is a cationic dye that can accumulate in the mitochondria. At low concentrations, it mainly exists as a monomer (monomer), and the emitted light is mainly green light (~ 525nm); at high concentrations, it can. Aggregation is formed, and the emitted light is mainly red light (-590nm). The mitochondria themselves have a certain polarity, the outer membrane is negative, and the inner membrane is positive. The potential difference is regulated by Ca2 +, Na + and H + flow. When the mitochondria are in good condition, the intake of JC-1 is small, so the ratio of green light intensity / red light intensity exists mainly in the form of monomers in the mitochondria. When depolarization occurs in the mitochondria, the concentration of JC-1 in the mitochondria is relatively high, mostly in the form of polymers, and the ratio of green light intensity / red light intensity decreases. The green light intensity / red light intensity of JC-1 staining only depends on the membrane potential of mitochondria, and has nothing to do with the shape, volume and density of mitochondria, so it can better reflect the functional state of mitochondria. Because of the mitochondrial depolarization in the early stage of apoptosis, JC-1 staining was used to detect the early stage of apoptosis. The experimental method is as follows. JC-l staining is very simple. First, the finished product JC-1 can be made into a storage solution (1 ~ 5 mg / ml) with DMSO, stored at -20 ℃, and diluted with the culture solution to a final concentration of 10 ug / ml. For adherent cells, the culture solution can be directly discarded, and the staining solution can be directly added after rinsing the cells. After 10 to 30 minutes, observe the fluorescence microscope or confocal laser. When the mitochondria are in good condition, the cells are mainly green. When the red light signal is enhanced, the red and green overlap, mainly orange. When the staining is applied to suspended cells, it can also be detected by flow cytometry to collect the red / green signal intensity and calculate the intensity ratio. Calcein-AM (Calcein-AM): Calcein-AM itself is not a fluorescent molecule, but through the action of esterase in living cells, Calcein-AM can remove the AM group, and the resulting Calcein can emit strong green fluorescence (excitation: 490 nm, emission: 515 nm).
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