CN101750435A - Method for detecting early apoptosis with electrorotation technology - Google Patents
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Abstract
The invention provides a method for detecting early apoptosis with an electrorotation technology, which comprises the steps: (1) for cell to be detected, adding apoptosis inducing factor in the liquid medium of the cell to make the cell have apoptosis, (2) making the cell to be detected suspend on the liquid medium, and adding the liquid medium into an electrorotation detection cell, (3) adopting liquid medium which is the same as the liquid medium in the electrorotation detection cell, and perfusing the liquid medium in the electrorotation detection cell, (4) generating uniform electric field in the electrorotation detection cell to lead the cell to be detected to have electrorotation action, and (5) changing the frequency of the rotating electric field to detect the rotating speed of the cell to be detected under the corresponding condition, and obtaining an electrorotation spectrum. The invention adopts the electrorotation technology as a non-invasive method to detect the early apoptosis, ensures the accuracy of the detection, and in addition, can lead the object to be detected to keep activity, and can realize the continuous study of one batch or single body.
Description
Technical field
The present invention relates to answer the electricity consumption rotation technique to detect the method for the physiological change of cell generation apoptotic process, particularly detect the method that cell generation early apoptosis changes.
Background technology
Apoptosis is meant for keeping homeostasis, by the autonomous orderly phenomena of mortality of the cell of Gene Handling.It can remove the cell of excessive or being potentially dangerous property in the body timely and effectively, is used to keep the balance of all kinds cell number in the body.This balance is for promoting organ to form and to keep stablizing of organismic internal environment most important.So the significance for apoptosis research has been opened the new window that we study for cancer drug at it.But, cause cancer patient still can recur after the chemotherapy clinically because apoptosis control program defective, the cancer cell development of cancer cell go out the resistance to the action of a drug.Present research has been found that the cancer cell of number of different types all can restore from apoptotic process, prove that cancer cell may utilize certain unknown escape mechanism to make and own renew work in that chemotherapy is follow-up.Therefore, pair cell early apoptosis and recuperation Study on Mechanism thereof will be significant for from now on antitumor work and medicament research and development aspect, so a kind ofly can make things convenient for, the means of the monitoring cellular physiological activity of non-intervention just seem very necessary.
Be mainly intervention property technology at apoptotic detection method at present, can be divided into following a few class:
1. use a certain target spot of dyestuff pair cell to dye, obtain corresponding information by fluorescence is measured, as using rhodamine 123 chromonema plastochondrias, when mitochondrial membrane potential descended, fluorescence weakened gradually; Perhaps use the nuclear staining of propidium iodide pair cell, in the apoptotic process, nucleus is wrapped in the apoptotic body, and with the process of apoptosis, cell within a cell nuclear reduces gradually, and fluorescence intensity reduces.General laser scanning co-focusing microscope or the flow cytometer of using of pair cell dyeing detects.
2.DNA segmentization is an apoptosis key character in late period.Common agarose gel electrophoresis can detect the segmentization of DNA.DNA extraction is come out, carry out electrophoresis, apoptotic cell will produce stair-stepping electrophoretic band, approximately is 180bp at interval.This method is more suitable for the many experiment of apoptotic cell quantity, but for small amounts of cells insufficient sensitivity then.At present dna segment detection method more commonly used is the TUNEL method, but its expense is also than electrophoresis height, and may have the false positive of non-viable non-apoptotic cell or other cell.
3. Apoptosis can make tenuigenin, nucleus and cell membrane that a series of biological chemistries and variation physically take place.Early stage at Apoptosis, cell expansion becomes circle, breaks off and break away from the back shrinkage with getting in touch of adjacent cells.In tenuigenin, endoplasmic reticulum swelling hydrops forms vacuole.In nucleus, chromatin aggegation gradually becomes crescent, is attached to the nuclear membrane periphery, and basophilla strengthens.The final cell karyorhexis is the fragment by the nuclear membrane parcel.On cell membrane, the cell node is no longer continuous, and cell membrane becomes more active and then caves in.It is the apoptotic body that is wrapped up cellular content by cell membrane that these variations all will cause lysis.Can use the immobile liquid fixed cell at present, by utilizing the shrinkage of electron microscope observation cell interior nucleus, perhaps apoptosis stage of being in of judgement cell such as surface such as cell membrane apoptotic body.
Above describedly be present generally popular detection apoptosis technology, but still more or less there are some problems in they.For numerous means wherein, the difference of research object may cause result of study and truth substantial deviation.The acceptor that a problem at first is exactly different research objects or the expression difference of albumen.Test such as Annexin V, different cells are expressed the difference of quantity owing to phosphatidylserine on the cell membrane own, the result that may cause is exactly, be applied in a kind of method on the cell of high expressed and can obtain satisfied result, but in apoptotic process, then may can't see obvious variation at all for those low cells of expressing phosphatidylserines.Generally acknowledge the caspase family protein that in apoptotic process, plays an important role for another example at present, expression at the cell interior of different genera different tissues is also completely different, lack functional caspase-3 albumen as breast cancer cell line MCF-7, so when using the DEVD substrate to detect the caspase-3 activity, the apoptosis situation may be underestimated.
It is different often that the another one problem is exactly different biochemical events or active time point, and a lot of albumen or receptor expression can reach peak value rapidly at point sometime, descend then.If miss this time period, will cause conclusion gross error.Such as after using anti-Fas monoclonal antibody to handle the Jurkat cell, the caspase-8 activity began to increase sharply in processing in back 2 hours, just reached peak value in 3 hours, and after this activity descends gradually.If but when detecting the caspase-8 activity of 1 hour and 7 hours solvent control groups and anti-Fas monoclonal antibody processed group relatively, thereby then can think and draw the result that this processing mode can cell death inducing a little less than signal too, cause the conclusion mistake.
Except above two problems, sixty-four dollar question is, detection means at present commonly used without exception for measured object intervention is arranged, promptly by extra one or more materials that add, measured object is taken place can be detected with optics or other means, chemistry or biochemical variation such as dyeing or cracking, fixed cell, obtains corresponding results by instrument detecting then.In whole testing process, cell is because detection means is limit, and the adding of the material factor that it is necessary can cause the physiological variation more or less of tested body itself, even death.Adopt such means research in addition,, just can't continue to observe their variations in the process in the back, understand a process, just can only adopt many batches sample to reach each stage of process, observation respectively respectively sample in case there is material such as dyestuff to get involved.
Comprise that the electrodynamics technology of electricity in being rotated in is a kind of analysis means of emerging, non-invasi, simple and flexible.For electric rotation technique, its sharpest edges are its non-invasis, only apply electric field by the outside, calculate the electric-physiology parameter that the cell rotating speed just can obtain being correlated with under the different frequency, thereby infer that cell is in different physiological statuss.At present existing a small amount of bibliographical information is all the dielectrophoresis of one of electrodynamics technology as a kind of technology of distinguishing normal cell and apoptotic cell.And, its variation when being used to detect early apoptosis of cells of bibliographical information is not arranged yet in electricity rotation field.If cell dielectric changes in the early apoptosis process, we are by the non-invasi of electrodynamics technology such as electricity rotation so, not needing to make cell to change just can make a distinction normal cell and apoptosis individual cells in various degree, keep activity then to same individual next step research that continues to cultivate or carry out, this method also is convenient to adopt the whole process that detects apoptosis with batch to be tested.
Summary of the invention
The invention provides a kind of method of detecting early apoptosis with electrorotation technology, when detecting Apoptosis in the prior art to solve because the acceptor of different research objects or the expression difference of albumen, or, cause the detection of prior art inaccurate because the time point of different biochemical events or activity is often different; And existing detection means has intervention for measured object, can't continue to observe the measured object variation in the process in the back.
For solving the problems of the technologies described above, the method for a kind of detecting early apoptosis with electrorotation technology of the present invention comprises the steps:
(1) for cell to be measured, in its residing liquid medium, add apoptosis inducing factor, make cell generation apoptosis to be measured;
(2) with cell suspension to be measured in liquid medium, join in the middle of the electricity rotation detection cell;
(3) adopt and the described electric identical liquid medium of liquid medium in the detection cell that rotates, perfusion in electricity rotation pond;
(4) in electricity rotation pond, produce uniform rotating electric field, make cell to be measured that electric circling behavior take place;
(5) frequency of change rotating electric field to measure the rotating speed of cell under corresponding conditions, obtains electricity rotation spectrogram.
Wherein, the described apoptosis inducing factor in the step (1) is TNF, TGF, glucocorticoid, virus, bacterium, free radical or chemotherapeutics.
Wherein, the perfusion rate described in the step (3) is 0.1mL/min-0.5mL/min.
Wherein, described perfusion rate is 0.25mL/min.
Wherein, producing uniform rotating electric field described in the step (4) is: applying four tunnel sinusoidal electric signals that phase place differs 90 ° successively successively on equally distributed four platinum electrodes around the electric wheel measuring pond.
Wherein, the amplitude range of the voltage of described sinusoidal electric signals is 1V-20V.
Wherein, the amplitude range of the voltage of described sinusoidal electric signals is 6V-10V.
Wherein, the frequency range of rotating electric field is 1kHz-1GHz in the step (5).
Wherein, the conductivity of described liquid medium is 1 μ S/cm-2mS/cm.
Wherein, the conductivity of described liquid medium is 200 μ S/cm-800 μ S/cm.
The present invention makes the electricity consumption rotation technique detect the early apoptosis of cell as a kind of non-intervention means, can distinguish out normal cell and apoptotic cell in the period that early apoptosis of cells can reverse, be not subjected to the expression difference of the acceptor or the albumen of different research objects, or different biochemical events or the active different influence of time point, guaranteed the accuracy that detects.And detection method of the present invention makes measured object can keep active, makes measured object can continue to cultivate or carry out next step research.This method applicable scope comprises unicellular organism and vegetable cell and zooblast, is particularly suitable for mammiferous normal cell and tumour cell.Can accomplish single batch or single individuality are studied continuously with respect to the other technologies of present research apoptosis, and experimental results show that the mensuration process is to measured object physiologically active did not influence.
Description of drawings
Fig. 1 is used to detect the electrorotation chip of early apoptosis of cells and the structure diagram of perfusion device for the present invention;
Fig. 2 is the electricity rotation collection of illustrative plates behind the cell death inducing different time;
Fig. 3 a is the coloration result figure of active oxygen dyestuff DCFH-DA to the cell of different apoptosis time;
Fig. 3 b is the comparison of cell at different apoptosis reactive oxygen species under the time;
Fig. 4 a is the figure as a result that Annexin V-FITC/PI kit is handled the cell of different apoptosis times;
Fig. 4 b is that cell is in the comparison of different apoptosis time points through Annexin V-FITC dyeing back fluorescence level;
Fig. 5 is the aspect graph of cell under the scanning electron microscope of different apoptosis time points.
Description of reference numerals
1-perfusion water inlet kapillary; 2-perfusion water outlet kapillary; The 3-platinum electrode; The 4-base plate; The 5-detection cell;
Embodiment
For characteristics of the present invention can be understood better, below will enumerate preferred embodiment and also be elaborated in conjunction with the accompanying drawings.
The method of detecting early apoptosis with electrorotation technology of the present invention, its principle is: in apoptotic process, cell expansion becomes circle, microvillus reduces gradually even disappears on the cell membrane, the appearance of apoptotic body, the ion channel in cell membrane opening causes the interior potassium ion of cell to be lost in a large number, satellite phenomenons such as polyunsaturated fatty acid initiation lipid peroxidation in the active oxygen attack biological film phospholipid that rolls up all affect the form of cell to a great extent, dielectric properties with cell membrane and cell interior, the change of these factors is all related with the behavior existence of cell in the electricity rotation, makes and may utilize electric wheel measuring to detect the early apoptosis of cell as means.
The early stage cell expansion of apoptosis becomes round, cell surface microvilli fades away, potassium-channel is opened, to medium cell shrinkage, chromatin concentrate, the intact nuclear membrane disappearance, to the appearance of apoptotic body in late period, this a series of process all generally occurs in the apoptotic process of cell.Compared to other technology that detects a certain albumen or acceptor, the variation of answering the electricity consumption rotation technique to measure dielectric properties will have ubiquity more.Cell dielectric character also is a successional variation in whole apoptotic process in addition, being different from numerous acceptors and albumen may only express in a certain moment, the variation of cell dielectric nature parameters is a unidirectional increase or reduction, thereby has reduced the possibility of missing a certain incident in the apoptotic process.Therefore this ubiquity of cell dielectric character and continuity can be used as one of standard of differentiating the apoptosis degree.
In addition, for the electric rotation technique that arrives involved in the present invention, itself be to utilize moment of torsion of the inner generation of electric field inducing cell, make cell produce circling behavior.In the middle of this process, the detection of electricity rotation not pair cell produces nonvolatil material and gets involved, therefore can the pair cell physiologically active, factor such as inner protein expression impacts.Cell is after making the detection of electricity consumption rotation technique, and physiological status will be before just the same with detection.
Therefore electric rotation technique can be used as a kind of convenience, rapid, effective monitoring means, in the process of using drug-induced apoptosis, at any time monitor the degree of carrying out of apoptosis, because the non-invasi of electric rotation itself can determine to continue apoptosis-induced or termination with a collection of cell.After stopping drug effect, cell can continue to cultivate, and also can add other inducer of apoptosis, blocking agent, or utilize other technologies further to detect.Therefore for other technologies, electric rotation itself has very strong dirigibility.
A kind of electric rotation detecting that is used to detect early apoptosis of cells of the present invention, it comprises base plate 4, and base plate 4 is the electronic chip plate, and detection cell 5 is located on the base plate 4, is the tubular space of a solid.Four platinum electrodes 3 evenly distribute around the detection cell 5, promptly are affixed on the inwall of detection cell 5 in the mode of 90 ° of quadratures each other, and the distance that two relative electrodes are 3 is 400 μ m.
Platinum electrode 3 is " L " shape, during detection, when measured object density when detecting medium, measured object is sunken to the bottoms of detecting media in the detection cell 5, at this moment the direction of electrode 3 is slotting from top to bottom, its position as shown in fig. 1, above the long limit of " L " shape is positioned at.When measured object density when detecting medium, measured object floats over the tops of detecting media in the electricity rotation detection cell 5, at this moment the direction of electrode 3 is slotting from the bottom up, below promptly the long limit of " L " shape was positioned at, minor face still was affixed on pool wall.
Perfusion device comprises perfusion water inlet kapillary 1, perfusion water outlet kapillary 2 and external pump.Perfusion water inlet kapillary 1 stretches in the detection cell 5, by the effect of external pump the medium solution drainage is gone in the detection cell 5; Perfusion water outlet kapillary 2 stretches in the detection cell 5, is positioned at the position relative with perfusion water inlet kapillary 1, and perfusion water outlet kapillary 2 uses another external pump that medium solution is pumped detection cell 5, thereby guarantees to be always fresh detection medium solution in the detection cell 5.
When the particle proportion that detects was bigger than liquid medium, perfusion water inlet kapillary 1 was positioned at detection cell 5 tops, made the liquid medium that perfusion water inlet kapillary 1 flows out import detection cell 5 from detection cell 5 tops; When the particle proportion that detects than liquid medium hour, perfusion water inlet kapillary 1 is positioned at detection cell 5 belows, makes liquid medium that perfusion water inlet kapillary 1 flows out from detection cell 5 belows input detection cell 5.
When biologic grain is in the electricity rotation detection cell 5 thus, is not produced displacement and rotation, thereby make the temperature of biologic grain environment of living in and medium composition remain unanimity by the institute's disturbance of perfusion action effect.
Catheter diameter can be at 100 μ m between the 300 μ m, preferably 200 μ m.The flow velocity that medium pumps into electricity rotation pond can be controlled in 0.1mL/min between the 0.5mL/min, preferably 0.25mL/min.External pump can adopt common constant flow pump, peristaltic pump or syringe pump.
Signal generator is connected with base plate 4, signal generator produces sinusoidal signal, and the electronic chip by base plate 4 differs four tunnel phase places from 90 ° sinusoidal signal successively and is applied to successively on four platinum electrodes 3, makes to form three-dimensional three-dimensional electric field in electricity rotation detection cell 5.Signal generator puts on the amplitude range of voltage of the electric signal on the electrode 3 between 1V to 20V, being good between the 6V to 10V.
The method of a kind of detecting early apoptosis with electrorotation technology of the present invention adopts above-mentioned electric rotation detecting, and its step comprises:
(1) for cell to be measured, in its residing liquid medium, add apoptosis inducing factor, make cell generation apoptosis to be measured.
Promptly be when the cell normal condition, give one or more apoptosis inducing factor inducing cells and enter apoptotic state that the apoptosis induction time can be designed voluntarily according to document or actual experiment result.Apoptosis inducing factor can be TNF, TGF, glucocorticoid, virus, bacterium, free radical or chemotherapeutics.Tested cell can be unicellular organism such as microorganism or protozoan, or the cell of higher organism such as vegetable cell or zooblast.
(2) at cell by after apoptosis-induced, cell suspension to be measured is had in the liquid medium of apoptosis inducing factor in this adding, move in the electricity rotation detection cell, and the conductivity of regulating this liquid medium.
(3) will rotate the liquid medium perfusion that liquid medium is identical in the detection cell with electricity by perfusion device goes in the electricity rotation pond.
(4) the uniform rotating electric field of generation in electricity rotation pond, and the frequency of change signal changes the frequency of rotating electric field, and the rotating speed under corresponding conditions of measuring cell obtains the electricity rotation and composes, and rotates the variation of spectrum by electricity and understands apoptotic degree.
The generation of rotating electric field uniformly can apply the electric signal that phase differential differs 90 ° successively by signal generator on four electrodes, central authorities produce the Rotating with Uniform electric field in electricity rotation pond, cell can rotate in rotating electric field, and its rotational speed is relevant with dielectric properties own.The scope of the electrical signal detection frequency that signal generator applies is between 1kHz to 1GHz, and that preferable is 10kHz to 20MHz.
When measuring cell electricity rotation spectrum, liquid medium can be chosen as the solution of monose such as glucose, fructose, galactose; The solution of disaccharide such as sucrose, lactose, maltose; The solution of polysaccharide such as starch, cellulose, pectin, sweet mellow wine; The solution of amino acid such as glycocoll, arginine, tryptophane, leucine etc.
Liquid medium also can be separated natural nutrient solutions such as liquid, lactalbumin hydrolysate or serum for tissue water.Or for synthesizing nutrient solution such as DMEM, RPMI1640, MEM, Medium 199, CMRL1066 or Ham ' s F-12; Balanced salt solution such as phosphate buffer, sodium chloride and Klorvess Liquid, Hanks liquid, EarleShi liquid or EagleShi liquid etc.
Medium conductivity can use the nutrient solution of phosphate buffer, sodium chloride, potassium chloride, Hanks liquid, EarleShi liquid, EagleShi liquid, DMEM, RPMI1640, MEM, Medium 199, CMRL1066 or Ham ' s F-12 to regulate, the range of adjustment of the conductivity of medium solution can be between 1 μ S/cm-2mS/cm, and preferably 200 μ S/cm are between the 800 μ S/cm.
Below enumerate the embodiment that HepG2 cell electricity consumption rotation technique after Ethanol Treatment detects its early apoptosis, with the method for explanation detecting early apoptosis with electrorotation technology of the present invention.When apoptosis takes place in other cells under the effect of other apoptosis inducing factors, identical among its detection method and the embodiment, only need to choose its appropriate liquid medium and apoptosis inducing factor according to different cells, and this choose be those skilled in the art should know.
Concrete, the human hepatoma HepG2 cell uses the DMEM nutrient solution that contains 10% hyclone to cultivate, and adds the penicillin of 100U/ml and the streptomysin of 100 μ g/ml in the nutrient solution.Cellular incubation is at 37 ℃, 5% CO
2Incubator in.Use the Ethanol Treatment cell, make cell generation apoptosis.Ethanol Treatment concentration is 500mM.In the time period of processing time 0h, 2h, 4h, 6h, 8h and 10h that the cell taking-up is also centrifugal respectively, remove supernatant.
Cell is resuspended in etc. in the mannitol solution (300mOs/kg) that oozes, and conductivity uses PBS to be adjusted to 360 μ S/cm.Each experiment uses liquid-transfering gun to draw in the extremely electric rotation of the cell suspension pond of 100 μ L, opens peristaltic pump simultaneously and carries out perfusion.
When cell after pond bottom is stable, on electrode, apply rotating electric field.Select the best 8V of being of voltage in the present embodiment, every decimal system frequency band detects four points between electric signal frequency 10kHz-20MHz.The electric circling behavior of cell is observed by inverted microscope, camera and the video frequency collection card used on the microscope carry out videograph, the rotational speed of cell is analyzed by software subsequently, the electricity rotation collection of illustrative plates of different apoptosis periods that obtain the HepG2 cell after Ethanol Treatment.
The electricity rotation collection of illustrative plates of accompanying drawing 2 is the HepG2 cell after the 500mM Ethanol Treatment different periods.Every spectral line is represented the mean value of 20 cells, and we can see between the electric rotational spectrums of different periods and changing obviously.6h and 10h significantly are lower than normal cell in the rotational speed of low-frequency range, and characteristic frequency moves to high frequency.Because electric rotational spectrum has reflected the dielectric properties of tested cell itself, so can draw the dielectricity of cell itself from electric rotational spectrum figure very big change has taken place, and then has detected the early apoptosis of cell.
For with the contrast of electric rotary detecting method, use active oxygen experiment, phosphatidylserine to expose experiment and scanning electron microscope and the HepG2 cell is handled the identical time period of back with the same manner detect.
The active oxygen experiment uses 2, and (DCFH-DA Sigma) detects 7-dichlorodihydrofluorescein diacetate.Cell uses behind the 500mM Ethanol Treatment different time with cold PBS cleaning twice, hatches 15min in 37 ℃ subsequently in final concentration is the PBS of 10 μ M DCFH-DA.On laser scanning co-focusing microscope, detect afterwards.Fluorescence intensity uses the burnt software of copolymerization to measure, and every group is detected 100 cells.Accompanying drawing 3 shows that HepG2 cell ROS level after the 500mM Ethanol Treatment raises gradually, during 10h the ROS level than the growth of control group level about 100%.The rising of ROS level is one of significant incident of early apoptosis of cells in the cell, and the active oxygen test experience has proved that cell is in the early apoptosis of cells stage at this moment.
Phosphatidylserine exposes experiment and uses Annexin V-FITC/PI apoptosisde tection kit kit to detect.Cell uses 500mM Ethanol Treatment different time, and the proposed steps according to manufacturer dyes subsequently, and detects on flow cytometer.Accompanying drawing 4 shows that the HepG2 cell is after the 500mM Ethanol Treatment, and the phosphatidylserine on the cell membrane internal membrane exposes gradually, combines with detecting apoptosis reagent A nnexin V-FITC, can detect cell fluorescence and strengthen gradually.During 10h, the fluorescence intensity ratio control group level of FITC has increased about 50%.Exposing of cell membrane phospholipid acyl serine is one of significant incident of early apoptosis of cells, and the test experience that phosphatidylserine exposes has proved that cell is in the early apoptosis of cells stage at this moment.
In scanning electron microscope experiment, the cell of untreated fish group and processed group is used the DMEM cleaning of serum-free, the mannitol solution 15min that oozes such as is resuspended in.Cell is next centrifugal to be fixed on 1.5h in 3% glutaraldehyde solution, uses PBS to clean the back with the fixing 1h of 1% osmic acid, and dehydration and freeze-drying are spent the night, and image is obtained in the detection of use electron microscope behind the metal spraying.Accompanying drawing 5 shows that the HepG2 cell is covered with a lot of flagellums on the cell when normal condition, and cell shape is normal circular.2h after the 500mM Ethanol Treatment, small part cell flagellum begin to disappear; The cell flagellum of half change nearly is sparse behind the 6h, and apoptotic body occurs on the small part cell; During 10h, most of cell surface is more smooth relatively, apoptotic body occurred on quite a few cell.Experiment on scanning electron microscope has shown the process of cell than the form change of early apoptosis intuitively.In electric rotation test the variation of electricity rotation spectrum also corresponding the variation of this cytomorphology.
As from the foregoing, the conclusion that draws of the method for detecting early apoptosis with electrorotation technology can access the support of using active oxygen experiment, phosphatidylserine to expose experiment and scanning electron microscope.
The present invention by electric rotation technique can non-intervention the pair cell normal condition or be subjected to the extraneous factor influence after enter apoptosis state detect.The physiological change that cell progresses into apoptotic state by normal condition can be significantly show in electricity rotation collection of illustrative plates and obtains.
The present invention makes the electricity consumption rotation technique detect the early apoptosis of cell as a kind of non-intervention means, can distinguish out normal cell and apoptotic cell in the period that early apoptosis of cells can reverse, be not subjected to the expression difference of the acceptor or the albumen of different research objects, or different biochemical events or the active different influence of time point, guaranteed the accuracy that detects.And detection method of the present invention makes measured object can keep active, makes measured object can continue to cultivate or carry out next step research.This method applicable scope comprises unicellular organism and vegetable cell and zooblast, is particularly suitable for mammiferous normal cell and tumour cell.Can accomplish single batch or single individuality are studied continuously with respect to the other technologies of present research apoptosis, and experimental results show that the mensuration process is to measured object physiologically active did not influence.
The above description of this invention is illustrative, and nonrestrictive, and those skilled in the art is understood, and can carry out many modifications, variation or equivalence to it within spirit that claim limits and scope, but they will fall within the scope of protection of the present invention all.
Claims (10)
1. the method for a detecting early apoptosis with electrorotation technology is characterized in that, comprises the steps:
(1) for cell to be measured, in its residing liquid medium, add apoptosis inducing factor, make cell generation apoptosis to be measured;
(2) with cell suspension to be measured in liquid medium, join in the middle of the electricity rotation detection cell;
(3) adopt and the described electric identical liquid medium of liquid medium in the detection cell that rotates, perfusion in electricity rotation pond;
(4) in electricity rotation pond, produce uniform rotating electric field, make cell to be measured that electric circling behavior take place;
(5) frequency of change rotating electric field to measure the rotating speed of cell under corresponding conditions, obtains electricity rotation spectrogram.
2. the method for detecting early apoptosis with electrorotation technology as claimed in claim 1, it is characterized in that the described apoptosis inducing factor in the step (1) is TNF, TGF, glucocorticoid, virus, bacterium, free radical or chemotherapeutics.
3. the method for detecting early apoptosis with electrorotation technology as claimed in claim 1 is characterized in that, the perfusion rate described in the step (3) is 0.1mL/min-0.5mL/min.
4. the method for detecting early apoptosis with electrorotation technology as claimed in claim 3 is characterized in that, described perfusion rate is 0.25mL/min.
5. the method for detecting early apoptosis with electrorotation technology as claimed in claim 1, it is characterized in that the uniform rotating electric field of generation described in the step (4) is: applying four tunnel sinusoidal electric signals that phase place differs 90 ° successively successively on equally distributed four platinum electrodes around the electric wheel measuring pond.
6. the method for detecting early apoptosis with electrorotation technology as claimed in claim 5 is characterized in that, the amplitude range of the voltage of described sinusoidal electric signals is 1V-20V.
7. the method for detecting early apoptosis with electrorotation technology as claimed in claim 6 is characterized in that, the amplitude range of the voltage of described sinusoidal electric signals is 6V-10V.
8. the method for detecting early apoptosis with electrorotation technology as claimed in claim 1 is characterized in that, the frequency range of rotating electric field is 1kHz-1GHz in the step (5).
9. the method for detecting early apoptosis with electrorotation technology as claimed in claim 1 is characterized in that, the conductivity of described liquid medium is 1 μ S/cm-2mS/cm.
10. the method for detecting early apoptosis with electrorotation technology as claimed in claim 9 is characterized in that, the conductivity of described liquid medium is 200 μ S/cm-800 μ S/cm.
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| CN104520436A (en) * | 2012-06-18 | 2015-04-15 | 约翰·迈尔斯·布鲁巴赫 | Microorganism evaluation system |
| US9644229B2 (en) | 2012-06-18 | 2017-05-09 | Sobru Solutions, Inc. | Microorganism evaluation system |
| CN104520436B (en) * | 2012-06-18 | 2017-07-28 | 索布鲁解决方案公司 | Microorganism evaluation system |
| US10748278B2 (en) | 2012-06-18 | 2020-08-18 | Sobru Solutions, Inc. | Organism evaluation system and method of use |
| US11446660B2 (en) | 2012-06-18 | 2022-09-20 | Scanlogx, Inc | Organism evaluation system and method of use |
| CN102879557A (en) * | 2012-10-11 | 2013-01-16 | 南京大学 | Microscopic sample fixing device used for plant electrophysiological experiments |
| CN104049010A (en) * | 2014-06-27 | 2014-09-17 | 中国科学院苏州生物医学工程技术研究所 | Method for detecting apoptosis degree of cells in solution to be tested |
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