Biological Analysis

Experimental principle and steps of PI single staining method

The principle of PI single staining is mainly based on the characteristic changes that occur at the cellular, subcellular and molecular levels during apoptosis. These changes include changes in the nucleus, changes in organelles, changes in cell membrane components and changes in cell morphology, among which changes in the nucleus are the most characteristic.

Introduction to PI

The fluorescent dye PI (propidium iodide) is a nuclear staining reagent that can stain DNA. It is often used for apoptosis detection. The full English name is Propidium Iodide. It is an ethidium bromide analog that 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 probes such as Calcein-AM or FDA to simultaneously stain live and dead cells. The excitation and emission wavelengths of the PI-DNA complex were 535 nm and 615 nm, respectively.

Appearance: reddish brown powder Storage conditions: -20℃

Experimental principle of PI single staining method

1. Changes in the nucleus: Due to the change in the nucleus of apoptotic cells, the dyeability of various chromosomal fluorescent dyes to the DNA of apoptotic cells is changed. Studies have shown that staining of fixed apoptotic cells with various chromosomal fluorescent dyes reduces DNA stainability. Many scholars regard this decrease in DNA stainability as one of the hallmarks of apoptotic cells.

2. Light scattering properties: Changes in the morphology of apoptotic cells affect their light scattering properties. On a flow cytometer, the forward scattered light is related to the size of the cell, while the side scattered light reflects the refraction of light in the cell and is related to the number of particles in the cell. During apoptosis, the cells shrink and the volume becomes smaller, so the forward scattered light decreases. This characteristic is often considered to be one of the characteristics of apoptotic cells. In addition, due to the degradation of chromosomes and the formation of nuclear rupture during apoptosis, intracellular particles often increase, so the side scattered light of apoptotic cells often increases. During cell necrosis, the forward scattered light increases due to cell swelling; side scattered light also increases during cell necrosis, so apoptotic cells and necrotic cells can be distinguished according to the forward scattered light and side scattered light. However, it should be noted that the reliability of judging apoptotic cells based on forward scattered light and side scattered light is greatly affected by the uniformity of the detected cell morphology and the ratio of nuclear to cytoplasmic. Therefore, in some lymphocyte apoptosis, the reliability of detecting apoptosis by light scattering characteristics is better, but in tumor cell apoptosis, its reliability is poor. The main advantage of detecting apoptotic cells based on light-scattering properties is that light-scattering properties can be combined with surface immunofluorescence analysis of cells to distinguish lymphocyte subtypes that undergo selective apoptosis after these special treatments. It can also be used for the classification of living cells.

PI Single Staining Reagents and Instruments

1, PBS solution;
2, PI staining solution: Dissolve PI in PBS (pH7.4), the final concentration is 100ug/ml. Store in a brown bottle at 4°C in the dark.
3,70% ethanol 4,400 mesh sieve 5, flow cytometer

PI single staining experimental steps

1. Collect cells {about (1~5)×106 cells/mL}, centrifuge at 500~1000 r/min for 5min, and discard the culture medium.
2. Wash once with 3ml PBS.
3. Centrifuge to remove PBS, add ice-cold 70% ethanol for fixation, 4°C for 1-2 hours.
4. Centrifuge to discard the fixative and resuspend in 3ml PBS for 5min.
5. Filter through a 400-mesh sieve once, centrifuge at 500-1000 r/min for 5 min, and discard the PBS.
6. Stain with 1ml PI staining solution, and protect from light at 4°C for 30min.
7. Flow cytometry detection: PI is excited by argon ions for fluorescence, the wavelength of the laser light wave is 488 nm, and the wavelength of the emitted light wave is greater than 630 nm, producing red fluorescence to analyze the histogram of the fluorescence intensity of PI, and also to analyze the scatter of the forward scattered light to the side scattered light. picture.
8. Judgment of results: On the scatter plot or topographic map of forward scattered light versus side scattered light, compared with normal cells, apoptotic cells have lower forward scattered light, while side scattered light can be high or low, which is related to the type of cells ; When analyzing the histogram of PI fluorescence, first use the gate technique to exclude double or aggregated cells and cell debris that emit weak fluorescence. On the histogram of PI fluorescence, apoptotic cells appear one-two-two before G1/G0 phase ploidy peak. For example, if the fluorescence intensity at the position of G1/G0 phase is 1.0, the fluorescence intensity of the hypodiploid peak of a typical apoptotic cell sample is 0.45, and the PI fluorescence intensity of red blood cells of chicken and salmon can be used as a reference standard. 0.35 and 0.7, respectively, ensure that in between are not cell debris but intact cells.

Note: During apoptosis, the decrease in DNA stainability is considered to be one of the hallmarks of apoptotic cells, but this decrease in DNA stainability may also be due to a decrease in DNA content, or due to changes in DNA structure. It is caused by changes in its ability to bind to dyes. Care should be taken when analyzing the results.