CN101498645B - Method for single-cell continuous flow capillary electrophoresis dual-wavelength cell activity detection - Google Patents
Method for single-cell continuous flow capillary electrophoresis dual-wavelength cell activity detection Download PDFInfo
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- CN101498645B CN101498645B CN2009100793229A CN200910079322A CN101498645B CN 101498645 B CN101498645 B CN 101498645B CN 2009100793229 A CN2009100793229 A CN 2009100793229A CN 200910079322 A CN200910079322 A CN 200910079322A CN 101498645 B CN101498645 B CN 101498645B
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Abstract
The invention relates to a method for detecting cell activity with the unicellular continuous stream capillary electrophoresis technology under dual waves, belonging to the field of cytobiology. The method comprises the steps of dyeing cells to be detected and fixing the cells, detecting the fixed cells with a capillary electrophoresis instrument under dual waves, detecting the light absorption value of each cell under ultraviolet wave and visible wave, and representing the activity of the cell with the specific value of the ultraviolet wave absorption value and the visible wave absorption value. The invention has the advantages of rapidness, convenience, low cost and quantitative cell activity detection and can be used for detecting the cell activity for the cytobiology, the cytotoxicology, the drug screening and research, and the like.
Description
Technical field
The present invention is the method for single-cell continuous flow capillary electrophoresis dual-wavelength cell activity detection, belongs to the cell biology field.
Background technology
Cytoactive detects and cell concentration is determined at cell biology, cell toxicology, and fields such as drug screening and development are to use important techniques and means very widely, has important use and is worth.
Conventional cytoactive detects and mainly contains decoration method and mtt assay.Decoration method is to enter intracellular amount according to dyestuff to come the active strong and weak of characterize cells membrane permeability and then reacting cells.Thereby decoration method normally adopts dyestuff pair cell dyeing backs such as trypan blue or PI to provide the qualitative of cytoactive and sxemiquantitative conclusion by the number and the cell dyeing degree of cell in microscope visual inspection and the complicate statistics certain volume cell solution, personal error due to this visual inspection dyeing depth is very big, and the value of the dye levels of each cell can not be provided.And also there is bigger personal error in the method for complicate statistics cell number.Mtt assay is MTT (tetramethyl azo azoles salt) reagent can be reduced to purple crystal by yellow and be deposited on principle in the cell according to the succinate dehydrogenase in the living cells, detects the average activity that amount that colony's cell produces purple crystal reflects all cells in the cell solution by colourimetry.Ben is that mtt assay only can detect the average activity of whole cell colony, and can't judge the activity of individual cells.
Cell count is a cell biology, microbiology, the statistics and the characterizing method of the extremely important and requisite cell of research fields such as molecular biology and toxicology " amount ".Conventional method for cell count is to use cell counter and microscope.Its basic skills is: the preparation cell suspension, drop in tally (special thick slide, counting chamber is arranged on it, have vertical element that counting chamber is divided into many lattices at the bottom of the pond) on, covered, at microscopically one or more cells in the some lattices of direct census (known volume) with the naked eye, so just can estimate the quantity or the quantity ratio of these cells in the unit volume cell suspension then.In order to estimate accurately, need counting to be no less than till 100 cells.Total cellular score in the count plate four big lattice is carried out statistical treatment at plaid matching inner cell line ball, only counts left side and top as the line ball cell.Be calculated as follows then: cell number/ml=(4 * big lattice total cellular score/4) * 10000.This method of counting needs preceding preliminary work of loaded down with trivial details counting and cautious manual counting, and personal error is very big, inefficiency.Present commercial automatic cell counter mainly is the hemocytometer counting apparatus that is used for blood analysis, and its principle is cell solution to be suspended in contain in the electrolytical solution.Under the voltage effect, suspension cell granule can pass through specific aperture in the solution pool at random in electric field, and causes that the resistance of aperture detection place or light scattering change.Change by the resistance or the light scattering of measuring the aperture place, carry out the cell count in the solution pool.This method is not suitable for the counting of cell concentration when low, and the commercial apparatus price is more expensive.
Modern cell quantitative detection means has the flow cytometer that occurs the seventies.Its current collection, computing machine, laser, fluid technique are one, are that current function is the strongest, state-of-the-art cell quantitative instrument.The whole world has 6 tame instrument production firms approximately.But up to ten thousand cells of flow cytometer high speed analysis, and have powerful analytic function, can from a cell, record a plurality of parameters simultaneously, the report of existing about 20～30 kinds of application functions wherein also comprises cytoactive and Cytometric detection at present.But because flow cytometer costs an arm and a leg experimental cost height, operative technique requirement height.Its instrument is bought and is used non-scientific research and the medical institutions that generally are engaged in biological study to bear.Therefore, calculate, a kind of simple, cheap, accurate and practical analytical approach need be provided at conventional sense index-cytoactive analysis, cell count and the concentration of cell.
Capillary Electrophoresis is modern trace analysis.Clear superiorities such as it has efficiently, and is quick, and instrument cost and experimental expenses are low, the present invention has set up the capillary electrophoretic analysis method that cytoactive detects and cell concentration calculates.The dual wavelength cytoactive analytical approach of being set up makes it accurately be better than the normal dyeing method aspect the judgement in cytoactive, carries out the active colony's cytoactive analysis that then is better than mtt assay that detects one by one by pair cell.The Capillary Electrophoresis cell concentration is measured cell count is prepared, but valueization, and reduced personal error.Capillary Electrophoresis cytoactive detection of being set up and cell concentration computing method are in instrument cost, and experimental cost and operative technique requirement aspect more are better than flow cytometer.In addition, because cell activity changes, conventional cell analysis is limited by cell holding time and restriction analysis time, and among the present invention cell sample after immobilization is handled, stable in properties, be not subjected to the influence of sample retention time and analysis time, therefore greatly facilitate the cytoactive analysis, for the batch samples analysis provides reliable method.
Existing in recent years report is applied to the Apoptosis status detection with Capillary Electrophoresis.For example: 1) directly intact cell is carried out capillary zone electrophoresis.Because normal cell has different specific charges with apoptotic cell, thereby can be separated from each other in electrophoresis process, two cell peaks occur and just can judge phenomena of apoptosis, the area by two peaks is than the apoptosis degree that can judge cell; 2) by after extracting intracellular DNA, adopt Capillary Electrophoresis that dna fragmentation is carried out electrophoretic analysis again, judge whereby whether cell apoptosis takes place; 3) represent apoptotic degree by the activity that detects intracellular caspase-3 albumen.Above method 1 be with micro-cell solution as sample, go out the peak position difference according to the different conditions cell, carry out Apoptosis whether evaluation.Method 2 all is during according to different cell state with 3, and the change of properties of intracellular big molecule such as nucleic acid or specified protein is carried out Apoptosis whether evaluation.
At present, Capillary Electrophoresis being used for the calculating of cell activity detection by quantitative and cell concentration does not all appear in the newspapers as yet.The present invention utilizes capillary electrophoresis technique, in conjunction with cell trypan blue colouring method, mode with unicellular continuous sample introduction, make cell in the solution under electroosmotic flow drives by kapillary and under ultraviolet/visible dual wavelength, detect the light absorption value of cell one by one, realize normal cell by dual wavelength ratio, the computing method of cell mortality have been set up in cell activity quantitative test after dead cell and the drug treating.The present invention utilizes capillary electrophoresis technique, in the mode of unicellular continuous sample introduction, make in the solution cell under electroosmotic flow drives, detect cell one by one by kapillary and under ultraviolet wavelength, represent cell number with detected cell peak number, set up the quantitative calculation method of cell concentration
Summary of the invention
The objective of the invention is to have personal error and can not carry out unicellular detection by quantitative and problems such as cytoactive quantitative test and flow cytometer cost height in the method for solution routine measurement cytoactive for a kind of method of single-cell continuous flow capillary electrophoresis dual-wavelength cell activity detection is provided.
For achieving the above object, the technical solution used in the present invention is:
1) with cell dyeing to be measured, centrifugal, remove supernatant, clean cell with phosphate buffer (PBS).Repeated centrifugation is cleaned cell more than 3 times, with extracellular residual dyestuff flush away.Add cell fixation liquid in the cell precipitation after cleaning, carry out cell fixation, the set time is more than 1 hour; Behind the cell fixation, clean cell more than twice with PBS again, remove remaining immobile liquid, the cell of being fixed.By the cell after the immobilization being carried out sonicated 5 seconds～10 seconds, can destroy the cell aggregation phenomenon.
Wherein: coloring agent is the coloring agent that cytoactive detects usefulness; Cell fixation liquid can be mass percent in the formalin more than 10% or mass percent at the paraformaldehyde solution more than 4%; The volume ratio of cell precipitation volume and immobile liquid is 1: 10～1: 20.
2) immobilized cell is configured to 10 with 20～100mM electrolyte buffer liquid
3～10
4The cell solution of individual/ml concentration range places the capillary electrophoresis apparatus positive terminal that has been equipped with diode array detector to carry out electrophoresis; Under ultraviolet and visible dual wavelength, detect the absorbance value of each cell simultaneously respectively.Because of the different conditions cell all exist to absorb under ultraviolet light, absorb more by force and also under visible light, exist through the cell after the trypan blue dyeing, so with the dual wavelength absorption value of ultraviolet/as seen than this cell activity of value representation.
Wherein: damping fluid is a borax, phosphate, any one in the ammonium acetate; The capillary pipe length that capillary electrophoresis apparatus adopts is 30～60cm, internal diameter 10～100um; Electrophoretic voltage is 10～20kV, and the ultraviolet wavelength scope that adopts during detection is 200～300nm, and visible wavelength range is 570～590nm, 100～200 of each cell number scopes that detects.
Beneficial effect
1) the conventional cell biological method with artificial judgment activity behind the cell dyeing combines with modern trace analysis Capillary Electrophoresis, traditional cytoactive manual observation is judged obtained the value standard, has realized the quantitative test of cytoactive.
2) by measuring the information of each cytoactive, can obtain colony's cell activity information.When having overcome mtt assay mensuration colony cytoactive information, do not consider the deficiency of individual cells activated information.
3) having set up cell count is the capillary electrophoresis method that cell concentration is measured.
4) at the cell routine check and analysis, capillary electrophoresis apparatus is compared with flow cytometer, in instrument cost, and experimental cost, operative technique requires and experimentation is simple etc., and the aspect has advantage extremely significantly.
5) cell sample is because of after immobilization handles, and properties of samples is stable, is not subjected to the influence of sample retention time and analysis time, therefore greatly facilitates the cytoactive analysis, and the activity that helps large quantities of cell samples detects.Because the dyeing of the trypan blue of cell and other decoration method can be applicable to various types of cells, so the cytoactive analytical approach has good versatility.Method for cell count also has versatility for various cell analysis.
Description of drawings
Fig. 1 detects synoptic diagram for Capillary Electrophoresis is used for cytoactive, wherein: the electrophoretic buffer at 1-electrode place, the 2-positive and negative electrode, the 3-kapillary, the 4-diode array detector, 5-is by cell capillaceous, and EOF is the electroosmotic flow that produces in the Capillary Electrophoresis;
Fig. 2 is the result that unicellular continuous Capillary Electrophoresis 214nm of normal cell and 570nm dual wavelength detect;
Embodiment
Cultivate 8 bottles of Hela cells as sample (about 10
6Individual/bottle Hela cell).One bottle is used for 56 ℃ as normal cell, one bottle and handles and handled in 15 minutes, and obtaining dead cell, all the other 6 bottles of cells is 40,50,60,70 with drug concentration respectively, and the curcumin of 80 and 90 μ g/ml is handled, and obtains the cell to be measured of drug treating.To normal cell, dead cell and drug treating cell to be measured is to clean cell with phosphate buffer behind the 0.4% trypan blue dyeing 3min with mass percent respectively, with extracellular residual trypan blue dyestuff flush away.Concrete grammar is that pair cell solution is centrifugal through hydro-extractor 1000 commentaries on classics/min, removes supernatant, keeps cell.Add phosphate buffer again, centrifugal, repeat 3 times and clean.The mass percent that adds 200 μ l in the cell solution after cleaning is 10% formalin, carries out cell fixation, 12 hours set times.Behind the cell fixation, centrifugal again, usefulness phosphate buffer (PBS) cleans cell more than twice, removes remaining formaldehyde.With cell with 10
3～10
4The concentration of individual/ml is formulated in the 50mM borax soln, places anodal place electrophoresis.Deposition condition is: the 50mM borax is an electrolyte solution, 12kV voltage, long capillary tube 30cm.During electrophoresis, cell moves to negative pole with single celled state under the effect of electroosmotic flow, and in capillary end process diode array detector.The 214nm of 100 cells of record and the absorption value of 570nm wavelength.At first obtain Normocellular 570nm/214nm absorption value mean value (m
n) be 0.02, next is obtained through 56 ℃ and handles the dead cell sample (m that handled gained in 15 minutes
d) 570nm/214nm absorption value mean value be 0.21, again to 40,50, the cell sample to be measured of 60,70,80 and 90 μ g/ml drug treating is obtained 570nm/214nm absorption value mean value (m
t), its value is respectively 0.029,0.034, and 0.034,0.072,0.132,0.191.
Claims (4)
1. the method for a single-cell continuous flow capillary electrophoresis dual-wavelength cell activity detection is characterized in that the concrete steps of this method are as follows:
1) with cell trypan blue dyeing to be measured, centrifugal, remove supernatant, clean cell with phosphate buffer (PBS), repeated centrifugation is cleaned cell more than 3 times, with extracellular residual dyestuff flush away; Add cell fixation liquid in the cell precipitation after cleaning, carry out cell fixation, the set time is more than 1 hour; Behind the cell fixation, use phosphate buffer (PBS) to clean cell more than twice again, remove remaining immobile liquid, the cell of being fixed;
2) immobilized cell is configured to 10 with 20～100mM electrolyte buffer liquid
3～10
4The cell solution of individual/ml concentration range, place the capillary electrophoresis apparatus positive terminal that has been equipped with diode array detector to carry out electrophoresis, be to detect the absorbance value of each cell under the ultraviolet light of 200-300nm and the visible light dual wavelength that wavelength coverage is 570-590nm respectively simultaneously in wavelength coverage, with ultraviolet/visible dual wavelength absorption value than this cell activity of value representation.
2. the method for a kind of single-cell continuous flow capillary electrophoresis dual-wavelength cell activity detection as claimed in claim 1 is characterized in that: by the cell after the immobilization being carried out sonicated 5 seconds～10 seconds, can destroy the cell aggregation phenomenon.
3. the method for a kind of single-cell continuous flow capillary electrophoresis dual-wavelength cell activity detection as claimed in claim 1 is characterized in that: cell fixation liquid can be mass percent in the formalin more than 10% or mass percent at the paraformaldehyde solution more than 4%; The volume ratio of cell precipitation volume and immobile liquid is 1: 10～1: 20.
4. the method for a kind of single-cell continuous flow capillary electrophoresis dual-wavelength cell activity detection as claimed in claim 1, it is characterized in that: electrolyte buffer liquid is borax, phosphate, any one in the ammonium acetate; The capillary pipe length that capillary electrophoresis apparatus adopts is 30～60cm, internal diameter 10～100um; Electrophoretic voltage is 10～20kV, and the ultraviolet wavelength scope that adopts during detection is 200～300nm, and visible wavelength range is 570～590nm, 100～200 of each cell number scopes that detects.
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