Acridine Orange (AO), Ethidium Bromide (EB) and Propidium Iodide (PI), DAPI, Hoechst dye, EthD III, 7-AAD, RedDot1, 2, etc.
Dyes that penetrate the membrane:
AO: It has membrane permeability, can penetrate the cell membrane, and stain the nuclear DNA and RNA into green and red, respectively, so that the nucleus is green or yellow-green fluorescence.
EB: A highly sensitive fluorescent stain that excites an orange-red signal at the standard 302 nm.
DAPI: blue is a blue fluorescent dye that can penetrate the cell membrane. It can generate fluorescence more than 20 times stronger than DAPI itself when combined with DNA, but has no fluorescence enhancement when combined with single-stranded DNA. The staining sensitivity of DAPI to double-stranded DNA was higher than that of EB and PI, the fluorescence intensity was lower than that of Hoechst, but the photostability was higher than that of Hoechst.
Hoechst dyes: Blue A class of fluorescent dyes that label DNA in microscopy, the two most common are Hoechst33342 and Hoechst33258. Both dyes are excited at UV 350 nm and emit cyan/blue fluorescence near the emission maximum at 461 nm. Compared with DAPI, Hoechst33342 has an ethyl group added, which has stronger lipophilicity, so it can better penetrate the intact cell membrane and has less cytotoxicity.
RedDot 1 Dye: Red, ultra-nuclear selectivity, spectrally similar to DRAQ5 and DRAQ7. RedDot dyes can be excited by several common lasers and can fluoresce in the far infrared. The red near-infrared fluorescence of RedDot effectively distinguishes it from other commonly used fluorescent probes.
Impermeable Dyes:
PI: Differently passes through living cell membranes, but can pass through damaged cell membranes to stain nuclei. PI is the first choice as a red fluorescent counterstain. PI is often used in combination with fluorescent probes such as Calcein-AM or FDA to distinguish dead/live cells.
EthD III, 7-AAD, RedDot 2: cannot penetrate the cell membrane, but can distinguish necrotic cells; more suitable for the detection of apoptosis and necrosis experiments;
Nuclear fluorescent dye (PI DAPI Hoechst33342)
The nuclear fluorescent dye PI propidium iodide (PI) is a commonly used nuclear fluorescent dye. It cannot pass through the intact cell membrane, but PI can pass through the membranes of middle and late apoptotic cells and dead cells to stain the nucleus red. Under the excitation of green light (540nm wavelength), PI will be excited at 600nm (red light). Fluorescing brightly, PI bound to DNA in the nucleus fluoresces 20-30 times more intensely than unbound PI. 40016Propidium iodide(PI)100mg40017Propidium iodide, 1.0mg/1mL solution in water10mL
propidium iodide
English name: Propidium iodide, Propidium diiodide;
Molecular formula of PI: C27H34I2N4
Molecular weight: 668.39
Appearance: reddish brown powder
Application: DNA Staining
Staining principle: Propidium iodide (PI) is an analog of ethidium bromide, which emits red fluorescence upon intercalation in double-stranded DNA. Although PI cannot pass through living cell membranes, it can pass through damaged cell membranes and stain nuclei. PI is often used with fluorescent compounds such as Calcein-AM or FDA to simultaneously stain live and dead cells.
Spectral properties: The excitation and emission wavelengths of the PI-DNA complex are 535 nm and 615 nm, respectively.
Dyeing process:
1. Prepare a 10-50 μM solution of PI in PBS or appropriate buffer. a)
2. 1/10 volume of the medium volume of PI solution was added to the cell culture medium. b)
3. Cells were incubated at 37°C for 10-20 minutes.
4. Cells were washed twice with PBS or a suitable buffer.
5. Cells were observed with a fluorescence microscope with an excitation wavelength of 535 nm, an emission wavelength of 615 nm filters.
a) Since PI may be carcinogenic, handle with care.
b) The medium can also be replaced with 1/10 concentration of PI buffer.
Storage conditions: 4 ℃ protected from light It is irritating to the human body, please pay attention to proper protection
DAPI, 4′,6-diamidino-2-phenylindole (4′,6-diamidino-2-phenylindole), is a fluorescent dye that can bind to most of the A and T bases in DNA. Commonly used with fluorescence microscopy. Because DAPI penetrates intact cell membranes, it can be used for staining of live and fixed cells. Under fluorescence microscope observation, DAPI dye is excited by light of ultraviolet wavelength. When DAPI binds to double-stranded DNA, the maximum absorption wavelength is 358 nm, the maximum emission wavelength is 461 nm, and the wavelength range of its divergent light covers blue to turquoise. DAPI can also bind to RNA, but the fluorescence intensity generated is not as strong as that of DNA binding, and the wavelength range of its divergent light is about 400 nm. The divergent light of DAPI is blue, and the divergence wavelengths of DAPI and green fluorescent protein (GFP) or Texas Red dye (red fluorescent dye) only have a small overlap. Perform multiple fluorescent stainings on a single sample. DAPI can quickly enter living cells and combine with DNA, so DAPI is also regarded as a toxic substance and carcinogen to living organisms. In the process of use, attention should be paid to the handling procedures of operation and discard. DAPI is a fluorescent dye that can penetrate the cell membrane and bind to double-stranded DNA in the nucleus to play a labeling role. It can produce more than 20 times stronger fluorescence than DAPI itself. Compared with EB, the staining sensitivity to double-stranded DNA is higher. many times higher. Cells with blue fluorescence can be seen under the microscope. The efficiency of cell labeling by fluorescence microscope is high (almost 100%), and it has no toxic and side effects on living cells. DAPI staining is often used to detect apoptosis, and after staining, it can be observed by fluorescence microscopy or flow cytometry. DAPI is also commonly used for general nuclear staining and double-stranded DNA staining in some specific cases. The cells were stained with DAPI for 3 minutes after heat shock treatment, and the morphological changes of the nucleus could be seen under a fluorescence microscope.
