CN107290197A - A kind of living cells mitochondria quantity dyes detection method - Google Patents
A kind of living cells mitochondria quantity dyes detection method Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
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- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
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Abstract
Detection method is dyed the invention discloses a kind of living cells mitochondria quantity, is comprised 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%, is filtered, except degerming, to be kept in dark place；（2）Inoculating cell：Individual cells suspension is made into nutrient solution, is inoculated into about 2,000 10000, every hole cell in 96 orifice plates, per the μ L of pore volume 200 300；（3）Cultivate cell：Condition of culture is set according to cell type, cultivated 28 days；（4）Dyeing：Added per hole in the above-mentioned μ L of tetrazolium bromide stoste 20 30, cell culture incubator and continue to be incubated 3 5h；（5）Observation：It is careful to draw the μ L of culture supernatant 10 50 in hole, the fresh nutrient solutions of 200 300 μ L are added, and in observation blue-purple granule quantity under Powerful Light Microscope.The method that the inventive method carries out mitochondria dyeing counting using MTT, and existing a variety of colouring methods complement one another, and preferably can provide aided analysis method for cell science research work.
Description
Technical field
The present invention relates to a kind of cell biology colouring method, more particularly to a kind of new living cells mitochondria quantity dyeing
Detection method, belongs to biological technical field.
Background technology
Mitochondria（Mitochondrion）It is the organelle of manufacture energy in cell, is referred to as " the power of cell
house（Power plant）”.In addition to for cell supply energy, mitochondria also participates in such as cell differentiation, genetic system, information and passed
Pass and the process such as Apoptosis.
It is intracellular can be from hundreds of to thousands of, with cell physiological function and physiology shape containing mitochondrial number
State is relevant.Mitochondria is more sensitive to various inside and outside damages, and in cellular damage, the change of mitochondria quantity is most common changes
One of become.For example under physiological status the metabolism of Skin Cell be unable to do without the mitochondria worked orderly；In pathological state
Increasing for offline mitochondrial number is adaptation reaction to chronic cell injury etc..
Laser Scanning Confocal Microscope, flow cytometry analysis, the iron haematoxylin that current detection line mitochondrial number is used are dyed and strong
There is certain deficiency in the methods such as that green dyeing.Laser Scanning Confocal Microscope detection mitochondria needs first to prepare good sample, then
By showing accurate 3-D view inside individual cells, the knot of mitochondria quantity is obtained by observing 3-D view technology
Really, this make it that the operation machine that Laser Scanning Confocal Microscope is tested and analyzed is cumbersome, and consumption long time could complete sample
, easily there are partial deviations, influence mitochondria quantity statistics in detection, and the negligible amounts of sample detection observation.Fluidic cell
A series of attributes of the cell dispersion being suspended in liquid of instrument analysis detection and analysis, for the mitochondria quantity of cell interior
Analysis lacks detail resolution, thus the degree of accuracy is poor.
Garapa uniformly dyeing color, is a kind of laboratory staining technique, is mainly used in various amoebas and Giardia lamblia
The identification of trophozoite and packing, for the mitochondria lack of targeted of cell interior, causes the display resolution of dyeing relatively low, no
Beneficial to accurate counting statistics.
Strong that green dyeing, i.e. Janus green B dye liquors, be in the mitochondrial live cell dye of selectivity, mitochondria carefully
Born of the same parents' chromo-oxidase makes dyestuff keep the state of oxidation (i.e. colored state) in blue-green, and cytoplasm is close to colourless, with high power
Distribution of mitochondria and form are observed under microscope.It is good for that green dye toxicity stronger, and the price of reagent is higher.
So, existing colouring method is present such as：Cumbersome, instrument and equipment is expensive, detection sensitivity, accuracy not
The defects such as foot.Existing research need when different, it is necessary to dye the target of observation, object to be distinguished all such as
Environmental change and change, still need more different colouring methods to provide existing cell mitochondrial more research sides
Method, Research foundation is provided for different research purposes.
The content of the invention
It is an object of the invention to overcome cumbersome, detection present in prior art Mitochondria quantity detection method
The above-mentioned deficiency that speed is slow, result precision is more low dyes detection method there is provided a kind of new mitochondria.The new technique has
There is the technical method of simple and quick, sensitive, economic mitochondria dyeing, to be complementary to one another with traditional detection method, improve detection
The degree of accuracy.
In order to realize foregoing invention purpose, the invention provides following technical scheme：
A kind of living cells mitochondria quantity dyes detection method, comprises the following steps：
（1）Stoste is prepared：Tetrazolium bromide（MTT）It is dissolved in 100 ml phosphate buffer（PBS, pH=7.4）In, being configured to concentration is
0.3-1wt% solution, filters, except degerming, to be kept in dark place.
（2）Inoculating cell：Individual cells suspension is made into nutrient solution, 96 are inoculated into about 2000-10000, every hole cell
In orifice plate, per pore volume 200-300 μ L.The orifice plate of wherein model can be selected, such as 6,12,24,48 orifice plates, from 96 holes
Plate can cultivate more observation samples.
（3）Cultivate cell：Condition of culture is set according to cell type, cultivated 2-8 days, preferably 3-5 days.Culture 3-5 days with
Afterwards, cell confluency 60-80% in culture medium, the overall fusion ratio of cell is suitable, carries out being easier to see when dyeing observation
Examine mitochondrial overall quantity and situation of change.
（4）Dyeing：Added per hole in above-mentioned tetrazolium bromide MTT stostes 20-30 μ L, cell culture incubator and continue to be incubated 3-5h, it is excellent
Select 3.5-4.5 h.Suitable incubation time enables cell more fully to suck MTT, and is allowed to be gathered in mitochondrial internal
Or surrounding so that the result of follow-up dyeing observation is accurately and reliably.Incubation time is long, and cell becomes feeble and die, Observation of Mitochondria meter
Number result loses accuracy, and the directive significance for research is reduced.
（5）Observation：Culture supernatant 10-50 μ L in careful absorption hole, nutrient solution fresh addition 200-300 μ L, and in
Blue-purple granule quantity is observed under Powerful Light Microscope.
The living cells mitochondria quantity dyeing detection method of the present invention, using MTT dyeing processing living cells, first by cell
Cultivate in the range of the suitable proportion for being 60-80% to fusion rate, the notable color for showing bluish violet is then dyed by MTT.Cause
There is significant specific aim for the mitochondria of living cells for MTT, colour developing result clearly, while proper proportion fusion is thin
Born of the same parents' individual can facilitate optical microphotograph Microscopic observation statistical magnitude.
Its principle is tetrazolium bromide, i.e. 3- (4,5- dimethyl -2- thiazoles) -2,5- diphenyl bromination tetrazole tetrazolium bromides,【3-
(4,5- Dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide】, abbreviation MTT, tool
There is positive charge, through cell membrane and can reduce to form insoluble bluish violet by the succinate dehydrogenase in living cells mitochondria
Formazan（Formazan）, it is deposited on around cell mitochondrial, and dead cell is without this function.
The similar method that progress mitochondria detection is dyed by tetrazolium bromide MTT is at home and abroad there is no at present, this method
Using helping to overcome the shortcomings of traditional detection method, and it is complementary to one another with traditional detection method advantage, improves mitochondria quantity
Abnormal accuracy in detection.
The application for MTT mainly uses mitochondrial succinate dehydrogenase to reduce exogenous MTT in the prior art
Generate the bluish violet crystallization first a ceremonial jade-ladle, used in libation of water-insoluble（Formazan）And be deposited in living cells, then utilize dimethyl sulfoxide (DMSO)
（DMSO）First a ceremonial jade-ladle, used in libation is dissolved, solution is determined in specific wavelength（490nm）Absorbance value.According to the absorbance measured（OD values）, come
Judge living cells quantity, OD values are bigger, and cytoactive is stronger.Belong to the side that apparent whole cell survival rate is detected using MTT
Method, the first a ceremonial jade-ladle, used in libation amount only gone to have investigated in being dissolved in DMSO from overall angle characterizes overall fuzzy survival rate, not
By MTT application specific to the detection and analysis specifically designed for the mitochondria quantity in cell interior.The present invention is special by MTT colour developing
Property be applied to living cells mitochondria quantity and dye detection method, provide new departure for the detection of mitochondria quantity, widened line
The optional selection method of body quantity dyeing detection and analysis, has great importance.
The conventional technical means that condition of culture is those skilled in the art, different cell line trainings are set according to cell type
It may need to be applied to different culture mediums, nutrient composition when foster, may be referred to cell culture processes in the prior art
The suitable culture medium of selection.Cell culture condition is the cofactor of the implementation of the present invention.
Wherein, tetrazolium bromide solution is being prepared with effective in two weeks after.The thiophene of Fresh is found according to the research of inventor
The most accurately and reliably, the shelf-life of MTT solution is two weeks to the result of azoles indigo plant solution dyeing detection and analysis.In the mistake prepared and preserved
Cheng Zhong, container notes being kept in dark place, for example, can be encased with aluminium-foil paper and realize lucifuge.By the way that container is wrapped up into real with light screening material
Existing shaded effect, it is to avoid influence of the illumination for MTT solution.
Further, removed and degermed with 0.22 μm of membrane filtration when preparing tetrazolium bromide solution, dyeing is caused by filtering
Reagent high purity, impurity, the factor such as bacterium are reduced for dyeing the influence of later sample observation, while 0.22 μm of filter membrane
It is readily available, it is cheap and preferable for the filter effect of bacterium.
Further, the tetrazolium bromide solution prepared is preserved at a temperature of being placed on 0-8 DEG C, is preferably placed at 4 DEG C of environment guarantors
Deposit.Tetrazolium bromide solution carries out preserving the generation for advantageously reducing contamination precipitation in the environment of low temperature, can also suppress bacterium
Breeding, improves the colour developing efficiency of reagent.
Further, when inoculating cell is cultivated in step 2, according to the suitable cell culture of the type selecting of cell
Liquid.
Compared with prior art, beneficial effects of the present invention：
1. the method that the inventive method carries out mitochondria dyeing counting using MTT, and existing a variety of colouring methods are mended each other
Fill, preferably can provide aided analysis method for cell science research work, reduce because mitochondria Study on dyeing method pair
Problem of being obstructed is studied caused by particular case can not be adapted to very well.
2. the inventive method is mainly used in field of scientific study, contribute to the correlative study for mitochondrial disease（Including
Diagnosis and slow sick prognostic analysis management etc.）Objective basis is provided, the research and development application of Related product is beneficial to reduce social medical treatment
Cost.
3. the inventive method mainly can apply to life science neck for living cells mitochondria quantity dyeing detection method
Domain, including basic research and clinical research.It is expected that using the dyeing detection method every year about 2,000,000 times, traditional dyeing is being solved
While method is not enough, considerable economic benefit can be brought for manufacturing enterprise.
Brief description of the drawings：
Fig. 1 is that the mitochondria of cell interior under Powerful Light Microscope is dyed by MTT, shows dark color（Blueness）.
Embodiment
With reference to test example and embodiment, the present invention is described in further detail.But this should not be understood
Following embodiment is only limitted to for the scope of above-mentioned theme of the invention, it is all that this is belonged to based on the technology that present invention is realized
The scope of invention.
Embodiment 1]
The dyeing detection of living cells mitochondria quantity
Dyeing detection and analysis mitochondria quantity is carried out to fibroblast, idiographic flow is as follows.
1st, stoste is prepared：0.5 gram of tetrazolium bromide MTT, is dissolved in 100 ml phosphate buffer（PBS, pH=7.4）In, use
0.22 μm of membrane filtration is kept in dark place with except degerming, putting 4 DEG C（In two weeks effectively）.
2nd, inoculating cell：Fibroblast is made into individual cells suspension with nutrient solution, with every hole about 2000-10000
Cell is inoculated into 96 orifice plates, per the μ L of pore volume 200.
3rd, cell is cultivated：Condition of culture is set according to cell type, cultivated 4 days, cell confluency 60-80%.
4th, dye：Added per hole in the above-mentioned μ L of tetrazolium bromide MTT stostes 20, cell culture incubator and continue to be incubated 4 h.
5th, observe：It is careful to draw culture supernatant in hole, the fresh nutrient solutions of 200 μ L are added, and it is micro- in high power light
Microscopic observation blue-purple granule quantity.
As a result as shown in figure 1, the quantity of dark particle points out mitochondrial quantity in figure（Photochrome is shown as bluish violet
Particle）.By microphotograph counting statistics, the quantity of the composition fibrocyte mitochondrial is 600-2000 relative particles.
[embodiment 2]
The dyeing detection of living cells mitochondria quantity
Dyeing detection and analysis mitochondria quantity is carried out to fibroblast, idiographic flow is as follows.
1st, stoste is prepared：0.5 gram of tetrazolium bromide MTT, is dissolved in 100 mL phosphate buffer（PBS, pH=7.4）In, use
0.22 μm of membrane filtration is kept in dark place with except degerming, putting 3 DEG C.During preparation and preservation, container is encased with aluminium-foil paper
Lucifuge.
2nd, inoculating cell：Fibroblast is made into individual cells suspension with nutrient solution, with every hole about 2000-10000
Cell is inoculated into 96 orifice plates, per the μ L of pore volume 200.
3rd, cell is cultivated：37 DEG C, 5% CO2Culture amplification is carried out, is cultivated 5 days, cell confluency 65-80%.
4th, dye：Added per hole in the above-mentioned μ L of tetrazolium bromide MTT stostes 20, cell culture incubator and continue to be incubated 4 h.
5th, observe：It is careful to draw culture supernatant in hole, the fresh nutrient solutions of 200 μ L are added, and it is micro- in high power light
Microscopic observation blue-purple granule quantity.Mitochondrial quantity is 600-1900 relative particles.
Further, the inventive method is applied to fibroblast dyeing detection and analysis mitochondria quantity, into fiber finer
Born of the same parents are in 37 ± 0.5 DEG C, 5v% CO2Carry out culture amplification, incubation time 3-5 days.
[embodiment 3]
The dyeing detection of living cells mitochondria quantity
Dyeing detection and analysis mitochondria quantity is carried out to fibroblast, idiographic flow is as follows.
1st, stoste is prepared：0.5 gram of tetrazolium bromide MTT, is dissolved in 100 mL phosphate buffer（PBS, pH=7.4）In, use
0.22 μm of membrane filtration is kept in dark place with except degerming, putting 3 DEG C.During preparation and preservation, container is encased with aluminium-foil paper
Lucifuge.
2nd, inoculating cell：Fibroblast is made into individual cells suspension with nutrient solution, with every hole about 2000-10000
Cell is inoculated into 96 orifice plates, per the μ L of pore volume 200.
3rd, cell is cultivated：Condition of culture is set according to cell type, cultivated 4 days, cell confluency 60-80%.
4th, dye：Added per hole in the above-mentioned μ L of tetrazolium bromide MTT stostes 25, cell culture incubator and continue to be incubated 4 h.
5th, observe：It is careful to draw the μ L of culture supernatant 20 in hole, the fresh nutrient solutions of 250 μ L are added, and in high power light
Micro- Microscopic observation blue-purple granule quantity.Mitochondrial quantity is 700-2000 relative particles.
[embodiment 4]
The dyeing detection of living cells mitochondria quantity
Dyeing detection and analysis mitochondria quantity is carried out to fibroblast, idiographic flow is as follows.
1st, stoste is prepared：0.5 gram of tetrazolium bromide MTT, is dissolved in 100 mL phosphate buffer（PBS, pH=7.4）In, use
0.22 μm of membrane filtration is kept in dark place with except degerming, putting 3 DEG C.During preparation and preservation, container is encased with aluminium-foil paper
Lucifuge.
2nd, inoculating cell：Fibroblast is made into individual cells suspension with nutrient solution, with every hole about 2000-10000
Cell is inoculated into 48 orifice plates, per the μ L of pore volume 300.
3rd, cell is cultivated：37 DEG C, 5% CO2Culture amplification is carried out, is cultivated 3 days, cell confluency 60-80%.
4th, dye：Added per hole in the above-mentioned μ L of tetrazolium bromide MTT stostes 25, cell culture incubator and continue to be incubated 5 h.
5th, observe：It is careful to draw the μ L of culture supernatant 10 in hole, the fresh nutrient solutions of 200 μ L are added, and in high power light
Micro- Microscopic observation blue-purple granule quantity.Mitochondrial quantity is 800-1900 relative particles.
[embodiment 5]
The dyeing detection of living cells mitochondria quantity
Dyeing detection and analysis mitochondria quantity is carried out to fibroblast, idiographic flow is as follows.
1st, stoste is prepared：0.5 gram of tetrazolium bromide MTT, is dissolved in 100 mL phosphate buffer（PBS, pH=7.4）In, use
0.22 μm of membrane filtration is kept in dark place with except degerming, putting 4 DEG C.
2nd, inoculating cell：Individual cells suspension is made into nutrient solution, 96 are inoculated into about 2000-10000, every hole cell
In orifice plate, per the μ L of pore volume 200.
3rd, cell is cultivated：37 DEG C, 5% CO2Culture amplification is carried out, is cultivated 4 days, cell confluency 60-80%.
4th, dye：Added per hole in the above-mentioned μ L of tetrazolium bromide MTT stostes 30, cell culture incubator and continue to be incubated 5h.
5th, observe：It is careful to draw the μ L of culture supernatant 40 in hole, the fresh nutrient solutions of 300 μ L are added, and in high power light
Micro- Microscopic observation blue-purple granule quantity.Mitochondrial quantity is 650-2000 relative particles.
[comparative example 1]
Living cells mitochondria film potential is detected（Flow cytometer indirect analysis mitochondria quantity/function）
Using flow cytometry analysis fibroblast mitochondriaFilm potential, idiographic flow is as follows.
By 37 DEG C of nutrient solution of fibroblast, 5% CO2Culture amplification is carried out, is cultivated 3 days.Growth period of taking the logarithm is thin
Cell suspending liquid is made of PBS for born of the same parents, pancreatin EDTA digestion, and PBS is rinsed 3 times, uses JC-1（1mmol/L）Dyed, 37 DEG C
30min is balanced, the fluorescence intensity of flow cytomery cell calculates the ratio of green fluorescence and red fluorescence（Depolarize journey
Degree）.
As a result：The film potential depolarising degree ratio 0.8 of this group of cell mitochondrial obtained by flow cytometry analysis,
The film potential depolarising change of the cell mitochondrial can be analyzed by showing that JC-1 and flow cytometer coordinate, indirectly reflection mitochondria number
The change of amount/function, this method operating procedure is relatively complicated, and the cost of equipment is higher.
Note：JC-1 faces used time preparation, and -20 DEG C are kept in dark place, it is to avoid multigelation.
[comparative example 2]
Living cells mitochondria quantity is detected（It is good for that green dyeing）
Fibroblast is carried out to be good for that green dyeing detection and analysis mitochondria quantity, idiographic flow is as follows.
That green dye liquor is good for prepare：By 0.5g be good for that it is green be dissolved in 50mL physiological saline, be heated to 35-40 DEG C, make its molten
Solution.
It is made into individual cells suspension with nutrient solution, 37 DEG C, incubated 2 days of 5%CO2.Take the logarithm growth period cell, addition
Equivalent is good for that green dye liquor, after continuation is incubated, draws culture supernatant, adds the fresh nutrient solutions of 500 μ L（Cultivate in 96 holes
Plate）, and in observing the mitochondria relative populations that are colored under high power inverted microscope.
As a result：It is good for after that green dyeing, is the glaucous little particle of form of diverse, this group of cell under mitochondria microscope
Mitochondrial quantity is 750-1800 relative particles.This method is same with the method for the present patent application with simple to operate excellent
Point, can complement one another.
Note：Active cytochrome C oxidase can be that strong green oxidative color-developing, therefore this method operation is fast
Speed, to ensure the activity of cell.In addition, being good for, that green dyeing kinetics is fast, and fade is also fast, thus need to be grasped detection observation when
Machine.
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.
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