CN107290197A - A kind of living cells mitochondria quantity dyes detection method - Google Patents

Abstract

The invention discloses a method for staining and detecting the number of mitochondria in living cells, which includes the following steps: (1) stock solution preparation: thiazole blue is dissolved in 100 ml of phosphate buffer solution, prepared into a solution with a concentration of 0.3-1wt%, and filtered to remove Bacteria, kept away from light; (2) Cell inoculation: Make a single cell suspension with culture medium, and inoculate about 2000‑10000 cells per well into a 96-well plate, with a volume of 200‑300 μL per well; (3) Culture cells : Set the culture conditions according to the cell type and culture for 2‑8 days; (4) Staining: add 20‑30 μL of the above thiazolyl blue stock solution to each well, and continue to incubate in the cell incubator for 3‑5 hours; (5) Observation: Carefully pipette the culture in the well Add 200-300 μL of fresh culture medium to 10‑50 μL of supernatant, and observe the number of blue-purple particles under a high-power optical microscope. The method of the present invention utilizes MTT to carry out mitochondrial staining and counting method, complements each other with various existing staining methods, and can better provide an auxiliary analysis method for cell science research work.

Description

A method for staining and detecting the number of mitochondria in living cells

technical field

The invention relates to a cell biology staining method, in particular to a novel staining detection method for the number of mitochondria in living cells, and belongs to the field of biotechnology.

Background technique

Mitochondria (Mitochondrion) is the organelle that produces energy in the cell, known as the "powerhouse (power plant)" of the cell. In addition to supplying energy to cells, mitochondria are also involved in processes such as cell differentiation, genetic systems, information transmission, and apoptosis.

The number of mitochondria contained in a cell can range from hundreds to thousands, which is related to the physiological function and physiological state of the cell. Mitochondria are sensitive to various internal and external injuries, and changes in the number of mitochondria are one of the most common changes during cell damage. For example, under physiological conditions, the metabolism of skin cells is inseparable from normal functioning mitochondria; under pathological conditions, the increase in the number of mitochondria is an adaptive response to chronic cell damage.

Currently, confocal microscopy, flow cytometry analysis, iron hematoxylin staining and Jianna green staining methods used to detect the number of mitochondria have certain deficiencies. The detection of mitochondria by confocal microscopy requires a well-prepared sample, and then by displaying an accurate three-dimensional image inside a single cell, the results of the number of mitochondria can be obtained by observing the three-dimensional image technology, which makes the operation of confocal microscope detection and analysis cumbersome and consumes a lot of time It takes a long time to complete the detection of a sample, and the number of sample detection observations is small, which is prone to local deviations and affects the statistics of mitochondria. Flow cytometry analysis detects and analyzes a series of attributes of dispersed cells suspended in a liquid, and lacks detailed resolution for the analysis of the number of mitochondria inside the cells, so the accuracy is poor.

Iron hematoxylin staining, a laboratory staining technique, is mainly used for the identification of various amoeba and Giardia lamblia trophozoites and cysts. It lacks specificity for the mitochondria inside the cells, resulting in the display resolution of the staining Low, not conducive to accurate counting statistics.

Jianna green staining, that is, Janus green B staining solution, is a specific living cell dye for mitochondria. Cytochrome oxidase in mitochondria keeps the dye in an oxidized state (that is, colored state) and turns blue-green, while the cytoplasm is close to colorless. The distribution and shape of mitochondria were observed under a high-power microscope. Jianna green dye is more toxic, and the price of the reagent is higher.

Therefore, the existing staining methods have defects such as cumbersome operation, expensive instruments and equipment, insufficient detection sensitivity, and insufficient accuracy. Existing research requires that for different situations, the targets that need to be dyed and observed, and the objects to be distinguished may change with the change of the environment. More different staining methods are still needed to provide more research methods for existing cell mitochondria. Provide a research basis for different research purposes.

Contents of the invention

The purpose of the present invention is to overcome the above-mentioned deficiencies such as cumbersome operation, slow detection speed, and low accuracy of results in the detection method of mitochondrial quantity in the prior art, and provide a new detection method for mitochondrial staining. This new technology has a simple, fast, sensitive and economical method of mitochondrial staining, which complements traditional detection methods and improves detection accuracy.

In order to realize the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

A living cell mitochondrial quantity staining detection method, comprising the following steps:

(1) Preparation of stock solution: Dissolve thiazolium blue (MTT) in 100 ml of phosphate buffer solution (PBS, pH=7.4), prepare a solution with a concentration of 0.3-1wt%, filter to remove bacteria, and store in the dark.

(2) Cell inoculation: Prepare a single cell suspension with culture medium, inoculate about 2,000-10,000 cells per well into a 96-well plate, with a volume of 200-300 μL per well. You can choose the type of orifice plate, such as 6, 12, 24, 48-well plate, etc., and choose 96-well plate to cultivate more observation samples.

(3) Cultivate cells: set the culture conditions according to the cell type, and culture for 2-8 days, preferably 3-5 days. After 3-5 days of culture, the cell fusion rate in the medium is 60-80%, and the overall fusion ratio of the cells is appropriate. It is easier to observe the overall number and changes of mitochondria when performing staining observation.

(4) Staining: Add 20-30 μL of the above-mentioned thiazolium blue MTT stock solution to each well, and continue to incubate in the cell incubator for 3-5 hours, preferably 3.5-4.5 hours. Appropriate incubation time allows the cells to absorb MTT more fully and gather it inside or around the mitochondria, making the results of subsequent staining and observation accurate and reliable. If the incubation time is too long, the cells will die, the mitochondrial observation and counting results will lose accuracy, and the guiding significance for research will be reduced.

(5) Observation: Carefully pipette 10-50 μL of the culture supernatant in the well, add 200-300 μL of fresh culture solution, and observe the number of blue-purple particles under a high-power optical microscope.

The living cell mitochondria number staining detection method of the present invention uses MTT staining to treat the living cells, first cultures the cells to a fusion rate of 60-80% in an appropriate ratio range, and then displays a blue-purple color by MTT staining. Because MTT is significantly targeted to the mitochondria of living cells, the color development results are very obvious, and the appropriate proportion of fused individual cells can be conveniently observed and counted under an optical microscope.

Its principle is thiazole blue, that is, 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl bromide tetrazolium thiazole blue, [3-(4,5-Dimethylthiazol-2- yl) -2,5-diphenyl-2H-tetrazolium bromide], referred to as MTT, has a positive charge, can pass through the cell membrane and be reduced by succinate dehydrogenase in the mitochondria of living cells to form insoluble blue-purple formazan (Formazan), Deposits around the mitochondria of cells, which is not available in dead cells.

At present, there is no similar method for mitochondrial detection by thiazolium blue MTT staining at home and abroad. The use of this method helps to overcome the shortcomings of traditional detection methods, and complements the advantages of traditional detection methods to improve the detection accuracy of abnormal mitochondrial numbers.

The application of MTT in the prior art is mainly to use mitochondrial succinate dehydrogenase to reduce exogenous MTT to form water-insoluble blue-purple crystalline formazan (Formazan) and deposit it in living cells, and then use dimethyl sulfoxide to (DMSO) to dissolve formazan, and measure the light absorption value of the solution at a specific wavelength (490nm). According to the measured absorbance value (OD value), the number of living cells is judged. The larger the OD value, the stronger the cell activity. It belongs to the method of using MTT to detect the apparent overall cell survival rate. It only examines the amount of formazan dissolved in DMSO from an overall perspective to characterize the overall vague survival rate, and does not specifically apply MTT to specific cells. Detection and analysis of the number of mitochondria inside the cell. The invention applies the chromogenic property of MTT to the detection method of mitochondrial quantity staining in living cells, provides a new scheme for the detection of mitochondrial quantity, broadens the optional methods of mitochondrial quantity staining detection and analysis, and has important significance.

Setting the culture conditions according to the cell type is a routine technical means for those skilled in the art. When culturing different cell lines, different culture media and nutrient solution components may need to be applied. You can refer to the cell culture methods in the prior art to select a suitable culture medium . Cell culture conditions are ancillary factors in the practice of the invention.

Wherein, the thiazolyl blue solution is effective within two weeks after preparation. According to the inventor's research, it is found that the results of staining, detection and analysis of the freshly prepared thiazolium blue solution are the most accurate and reliable, and the shelf life of the MTT solution is two weeks. During the process of preparation and storage, the container should be protected from light, for example, it can be wrapped with aluminum foil to avoid light. The light-shielding effect is achieved by wrapping the container with light-shielding material to avoid the influence of light on the MTT solution.

Further, when preparing the thiazolium blue solution, use a 0.22 μm filter membrane to filter out bacteria. Through filtration, the purity of the dyed reagent is high, and the influence of impurities, bacteria and other factors on the observation of the sample after staining is reduced. At the same time, the 0.22 μm filter membrane is easy to obtain. The price is low, and the filtration effect on bacteria is better.

Further, the prepared thiazole blue solution is stored at a temperature of 0-8°C, preferably at 4°C. Storing the thiazolium blue solution in a low temperature environment is beneficial to reduce the generation of impurity precipitation, and can also inhibit the reproduction of bacteria and improve the color efficiency of the reagent.

Further, when inoculating cells for culturing in step 2, an appropriate cell culture medium is selected according to the type of cells.

Compared with prior art, the beneficial effect of the present invention:

1. The method of the present invention utilizes MTT to carry out the method for counting mitochondrial staining, which is complementary to the existing multiple staining methods, and can better provide auxiliary analysis methods for cell science research work, and reduce the number of research methods because mitochondrial staining cannot be used for specific situations. It is well adapted to the research obstruction problem caused by it.

2. The method of the present invention is mainly used in the field of scientific research, which helps to provide an objective basis for the research on mitochondrial diseases (including diagnosis and chronic disease prognosis analysis management, etc.), and the development and application of related products will help reduce social medical costs.

3. The method of the present invention can be mainly applied to the field of life sciences, including basic research and clinical research, for the staining detection method of the number of mitochondria in living cells. It is estimated that this dyeing detection method will be used about 2 million times per year, which can bring considerable economic benefits to production enterprises while solving the shortcomings of traditional dyeing methods.

Description of drawings:

Figure 1 shows the mitochondria inside the cell stained by MTT under a high-power optical microscope, showing a dark color (blue).

detailed description

The present invention will be further described in detail below in conjunction with test examples and specific embodiments. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

Example 1]

Staining detection of the number of mitochondria in living cells

The fibroblasts were stained to detect and analyze the number of mitochondria, and the specific process was as follows.

1. Stock solution preparation: 0.5 g of thiazolyl blue MTT, dissolved in 100 ml of phosphate buffer solution (PBS, pH=7.4), filtered through a 0.22 μm filter membrane to remove bacteria, stored at 4°C in the dark (effective within two weeks) .

2. Cell inoculation: Make a single cell suspension with culture medium for fibroblasts, inoculate about 2,000-10,000 cells per well into a 96-well plate, with a volume of 200 μL per well.

3. Culture cells: Set the culture conditions according to the cell type, culture for 4 days, and the cell fusion rate is 60-80%.

4. Staining: Add 20 μL of the above-mentioned thiazolium blue MTT stock solution to each well, and continue to incubate for 4 h in the cell culture incubator.

5. Observation: Carefully pipette the culture supernatant in the well, add 200 μL of fresh culture solution, and observe the number of blue-purple particles under a high-power optical microscope.

The results are shown in Figure 1, where the number of dark granules indicates the number of mitochondria (blue-purple granules in color photos). According to counting statistics of microscopic photos, the number of mitochondria in the fiber cells of this composition is 600-2000 relative particles.

[Example 2]

Staining detection of the number of mitochondria in living cells

The fibroblasts were stained to detect and analyze the number of mitochondria, and the specific process was as follows.

1. Stock solution preparation: Dissolve 0.5 g of thiazolium blue MTT in 100 mL of phosphate buffer solution (PBS, pH=7.4), filter through a 0.22 μm filter membrane to remove bacteria, and store at 3°C in the dark. During preparation and storage, the container was wrapped in aluminum foil to protect from light.

2. Cell inoculation: Make a single cell suspension with culture medium for fibroblasts, inoculate about 2,000-10,000 cells per well into a 96-well plate, with a volume of 200 μL per well.

3. Cultured cells: 37°C, 5% CO ₂ for culture expansion, cultured for 5 days, the cell fusion rate is 65-80%.

4. Staining: Add 20 μL of the above-mentioned thiazolium blue MTT stock solution to each well, and continue to incubate for 4 h in the cell culture incubator.

5. Observation: Carefully pipette the culture supernatant in the well, add 200 μL of fresh culture solution, and observe the number of blue-purple particles under a high-power optical microscope. The number of mitochondria is 600-1900 relative particles.

Further, the method of the present invention is applied to staining and analyzing the number of mitochondria in fibroblasts, and the fibroblasts are cultured and expanded at 37±0.5°C and 5v% CO ₂ for 3-5 days.

[Example 3]

Staining detection of the number of mitochondria in living cells

The fibroblasts were stained to detect and analyze the number of mitochondria, and the specific process was as follows.

1. Stock solution preparation: Dissolve 0.5 g of thiazolium blue MTT in 100 mL of phosphate buffer solution (PBS, pH=7.4), filter through a 0.22 μm filter membrane to remove bacteria, and store at 3°C in the dark. During preparation and storage, the container was wrapped in aluminum foil to protect from light.

2. Cell inoculation: Make a single cell suspension with culture medium for fibroblasts, inoculate about 2,000-10,000 cells per well into a 96-well plate, with a volume of 200 μL per well.

3. Culture cells: Set the culture conditions according to the cell type, culture for 4 days, and the cell fusion rate is 60-80%.

4. Staining: Add 25 μL of the above thiazolium blue MTT stock solution to each well, and continue to incubate for 4 h in the cell culture incubator.

5. Observation: Carefully pipette 20 μL of the culture supernatant in the well, add 250 μL of fresh culture solution, and observe the number of blue-purple particles under a high-power optical microscope. The number of mitochondria is 700-2000 relative particles.

[Example 4]

Staining detection of the number of mitochondria in living cells

The fibroblasts were stained to detect and analyze the number of mitochondria, and the specific process was as follows.

1. Stock solution preparation: Dissolve 0.5 g of thiazolium blue MTT in 100 mL of phosphate buffer solution (PBS, pH=7.4), filter through a 0.22 μm filter membrane to remove bacteria, and store at 3°C in the dark. During preparation and storage, the container was wrapped in aluminum foil to protect from light.

2. Cell inoculation: Make a single cell suspension with culture medium for fibroblasts, inoculate about 2,000-10,000 cells per well into a 48-well plate, with a volume of 300 μL per well.

3. Cultivate cells: 37°C, 5% CO ₂ for culture expansion, culture for 3 days, the cell fusion rate is 60-80%.

4. Staining: Add 25 μL of the above-mentioned thiazolium blue MTT stock solution to each well, and continue to incubate for 5 h in the cell culture incubator.

5. Observation: Carefully pipette 10 μL of the culture supernatant in the well, add 200 μL of fresh culture solution, and observe the number of blue-purple particles under a high-power optical microscope. The number of mitochondria is 800-1900 relative particles.

[Example 5]

Staining detection of the number of mitochondria in living cells

The fibroblasts were stained to detect and analyze the number of mitochondria, and the specific process was as follows.

1. Stock solution preparation: Dissolve 0.5 g of thiazolyl blue MTT in 100 mL of phosphate buffer solution (PBS, pH=7.4), filter through a 0.22 μm filter membrane to remove bacteria, and store at 4°C in the dark.

2. Cell inoculation: Make a single cell suspension with the culture medium, and inoculate about 2000-10000 cells per well into a 96-well plate with a volume of 200 μL per well.

3. Cultivate cells: 37°C, 5% CO ₂ for culture expansion, culture for 4 days, the cell fusion rate is 60-80%.

4. Staining: Add 30 μL of the above-mentioned thiazolium blue MTT stock solution to each well, and continue to incubate in the cell incubator for 5 hours.

5. Observation: Carefully pipette 40 μL of the culture supernatant in the well, add 300 μL of fresh culture solution, and observe the number of blue-purple particles under a high-power optical microscope. The number of mitochondria ranged from 650-2000 relative particles.

[Comparative Example 1]

Detection of mitochondrial membrane potential in living cells (indirect analysis of mitochondrial number/function by flow cytometry)

The mitochondrial membrane potential of fibroblasts was analyzed by flow cytometry, and the specific procedure was as follows.

The fibroblasts were cultured and expanded in the culture solution at 37°C and 5% CO ₂ for 3 days. Take the cells in the logarithmic growth phase, digest them with trypsin EDTA, make a cell suspension with PBS, rinse with PBS 3 times, stain with JC-1 (1mmol/L), equilibrate at 37°C for 30min, and detect the fluorescence of the cells by flow cytometry Intensity, the ratio of green fluorescence to red fluorescence (degree of depolarization) was calculated.

Results: The ratio of the membrane potential depolarization degree of mitochondria in this group of cells obtained by flow cytometry analysis was 0.8, indicating that the cooperation of JC-1 and flow cytometry can analyze the changes in membrane potential depolarization of mitochondria in the cells, which indirectly reflects the mitochondrial Quantity/function change, the operation steps of this method are relatively cumbersome, and the cost of equipment is relatively high.

Note: JC-1 is prepared just before use, and stored at -20°C in the dark, avoiding repeated freezing and thawing.

[Comparative example 2]

Detection of the number of mitochondria in living cells ( Jianna green staining )

Fibroblasts were stained with Jianna Green to detect and analyze the number of mitochondria, and the specific process was as follows.

Preparation of Jianna Green dye solution: Dissolve 0.5g Jiana Green in 50mL of normal saline, heat to 35-40°C to dissolve it.

The culture medium was used to make a single cell suspension, and cultured at a constant temperature of 37°C and 5% CO2 for 2 days. Take the cells in the logarithmic growth phase, add an equal amount of Kenna Green staining solution, continue to cultivate at a constant temperature, absorb the culture supernatant, add 500 μL of fresh culture solution (96-well culture plate), and observe the stained cells under a high-power inverted microscope. Relative number of mitochondria.

Results: After Kena Green staining, the mitochondria were small blue-green particles with various shapes under the microscope, and the number of mitochondria in this group was 750-1800 relative particles. This method and the method of the present application also have the advantage of simple operation and can complement each other.

Note: only the active cytochrome C oxidase can oxidize Jianna Green, so the method should be operated quickly to ensure the activity of the cells. In addition, the dyeing speed of Jianna Green is fast, and the color fades quickly, so it is necessary to grasp the timing of detection and observation.

Claims (9)

1. a kind of living cells mitochondria quantity dyes detection method, comprise the following steps：

（1）Stoste is prepared：Tetrazolium bromide is dissolved in 100 ml phosphate buffer, is configured to the solution that concentration is 0.3-1wt%, mistake Filter, except degerming, to be kept in dark place；

（2）Inoculating cell：Individual cells suspension is made into nutrient solution, 96 orifice plates are inoculated into about 2000-10000, every hole cell In, per pore volume 200-300 μ L；

（3）Cultivate cell：Condition of culture is set according to cell type, cultivated 2-8 days；

（4）Dyeing：Added per hole in above-mentioned tetrazolium bromide stoste 20-30 μ L, cell culture incubator and continue to be incubated 3-5h；

（5）Observation：It is careful to draw culture supernatant 10-50 μ L in hole, the fresh nutrient solutions of 200-300 μ L are added, and in high power Optical microphotograph Microscopic observation blue-purple granule quantity.

2. living cells mitochondria quantity as claimed in claim 1 dyes detection method, it is characterised in that step 1, tetrazolium bromide solution Preparing with effective in two weeks after.

3. living cells mitochondria quantity as claimed in claim 1 dyes detection method, it is characterised in that step 1, tetrazolium bromide solution During preparation and preservation, container notes lucifuge.

4. living cells mitochondria quantity as claimed in claim 1 dyes detection method, it is characterised in that step 1, tetrazolium bromide is prepared Removed and degermed with 0.22 μm of membrane filtration when solution.

5. living cells mitochondria quantity as claimed in claim 1 dyes detection method, it is characterised in that step 1, the thiophene prepared Azoles indigo plant solution is preserved at a temperature of being placed on 0-8 DEG C.

6. living cells mitochondria quantity as claimed in claim 5 dyes detection method, it is characterised in that step 1, the thiophene prepared Azoles indigo plant solution is placed on 4 DEG C of environment preservations.

7. living cells mitochondria quantity as claimed in claim 1 dyes detection method, it is characterised in that step 1, the phosphate PH of buffer=7.4.

8. living cells mitochondria quantity as claimed in claim 1 dyes detection method, it is characterised in that step 3, during cell culture Between be 3-5 days.

9. living cells mitochondria quantity as claimed in claim 1 dyes detection method, it is characterised in that step 4, cell culture incubator It is interior to continue to be incubated 3.5-4.5 h.