Cell staining method

Hoechst can cross the living cell membrane to bind to the nucleus (mainly apoptotic living cells) under ultraviolet light

The nucleus is stained blue. Hoechst staining the nucleus affects the observation of other fluorescence of the sample by confocal microscopy. hoechst has hoechest33342 and hoechst33258. The two hoechsts33258, hoechst33342 have little difference, but hoechst33342 has less toxic effects on cells. Therefore, in general, hoechsts33258 is used for cell fixation and then staining, while hoechst33342 can directly stain living cells!

PI (Propidium Iodide propidium iodide) staining

It is a nuclear staining reagent for DNA staining and is commonly used for apoptosis detection. Propidium Iodide (PI) is a nucleic acid dye (red) that does not penetrate intact cell membranes but is in apoptosis. In advanced cells and necrotic cells, due to the increase of cell membrane permeability, PI can nucleate the cell through the cell membrane. Observation of cultured cells with PI single staining can only indicate cell necrosis, not apoptosis (of course, late apoptosis) PI can also be colored). But if you just want to know the cell's death, rather than carefully distinguish between necrosis or apoptosis, then PI single staining is fine. But if you must identify the cell's apoptosis, then PI single staining is obviously not enough!

Annexin-v staining

In the early stage of apoptosis, the cell membrane marker changes. Among them, phosphatidylserine

(Annexin-V, PS) Eversion, Annexin-V specifically binds to its high affinity in the presence of Ca+. Thus, Annexin-v staining positive indicates that the cell is in an early apoptotic state. Annexin-V binds to different fluorescent antibodies. Flow cytometry, fluorescence microscopy, and confocal laser scanning microscopy can be used to detect apoptosis. Annexin V is fluorescently labeled with FITC; red fluorescent if 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, and the emitted light is mainly green light (~525nm). At high concentrations, it can form a polymer. (aggregation), the emitted light is mainly red light (-590nm). The mitochondria itself has a certain polarity

(polarization), the outer membrane is a negative electrode, and the inner membrane is a positive electrode. The potential difference is regulated by the Ca2+, Na+ and H+ flows. When the mitochondria is in a good state, the JC-1 is ingested in a small amount, and thus the ratio of the green light intensity/red light intensity in the form of a monomer in the mitochondria is high. When depolarization occurs in the mitochondria, the concentration of JC-1 in the mitochondria is high, mostly in the form of multimers, and the ratio of green light intensity to red light intensity decreases. The green light intensity/red light intensity of JC-1 staining depends only on the membrane potential of mitochondria, and is independent of the morphology, volume and density of mitochondria, thus better reflecting the functional state of mitochondria. JC-1 staining was used to detect the early onset of apoptosis due to the depolarization of mitochondria in the early stages of apoptosis. The experimental method is as follows.

JC-l staining is very simple. First, the finished JC-1 can be stored in DMSO as a stock solution (1 to 5 mg/ml), stored at -20 ° C, and diluted to a final concentration of 10 ug/ml with the culture solution. The adherent cells can be discarded directly, and the cells are directly added to the staining solution after rinsing, and observed under a fluorescence microscope or laser confocal after 10-30 min. When the mitochondria is in good condition, the cells are mainly green. When the red light signal is enhanced, the red and green are stacked, mainly orange. When the staining is applied to suspension cells, it can also be detected by flow cytometry, and the intensity of the red/green signal is collected, and the intensity ratio is calculated.

Calcein-AM:

Calcein-AM itself is not a fluorescent molecule, but Calcein-AM can remove the AM group by the action of esterase in living cells, and the resulting Calcein can emit strong green fluorescence (excitation: 490 nm, emission: 515 nm). Therefore Calcein-AM only stains live cells

Cell viability identification - trypan blue staining

principle:

When cells are damaged or die, trypan blue penetrates the denatured cell membrane and binds to the disintegrated DNA to color it. Living cells prevent the dye from entering the cell. Therefore, it is possible to identify dead cells and living cells.

Supplies:

1. 4% trypan blue mother liquor: Weigh 4g trypan blue, add a small amount of distilled water to grind, add double distilled water to 100ml, filter with filter paper, save at 4 degrees. when using it. Dilute to 0.4% with PBS. 2. Straw, blood cell counting plate, microscope

step:

1. Prepare a single cell suspension. And appropriate dilution (106 cells / ml) 2, staining: cell suspension and 0.4% trypan blue solution mixed with 9:1. 3. Counting: Counting live and dead cells separately using a counting plate within three minutes

Results statistics:

Under the microscope, the dead cells were stained light blue, while the living cells were rejected. Find cell viability according to the following formula:

Live cell rate (%) = total number of viable cells / (total number of viable cells + total number of dead cells) × 100

Note: When the trypan blue stained cells, the time should not be too long. Otherwise, some of the living cells will also be colored, which will interfere with the count.

Trypan blue staining principle

It is generally believed that the cell membrane loses its integrity and the cell can be considered dead. Trypan Blue is the most commonly used biostaining reagent for detecting cell membrane integrity. Healthy normal cells are able to reject trypan blue, while dead cells, membrane integrity is lost, permeability is increased, and cells can be stained blue by trypan blue. According to this principle, after staining with trypan blue, the cells can be counted by microscope, directly under the microscope or counted after photographing, so as to achieve a more accurate quantitative analysis of cell survival rate.

The principle of trypan blue dyeing to identify dead and living cells is described. The analysis of the long dyeing time is the reason why living cells are also stained.

The cell membrane of dead cells has no barrier effect, and trypan blue immediately enters the cell and appears blue. The viable cell membrane is selectively permeable, has little permeability to trypan blue, and enters cells slowly. If the staining time is too long, trypan blue may enter the cell by endocytosis or pinocytosis.