CN104964909A - Platelet transmembrane potential detection method - Google Patents
Platelet transmembrane potential detection method Download PDFInfo
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- CN104964909A CN104964909A CN201510280527.9A CN201510280527A CN104964909A CN 104964909 A CN104964909 A CN 104964909A CN 201510280527 A CN201510280527 A CN 201510280527A CN 104964909 A CN104964909 A CN 104964909A
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Abstract
The present invention relates to a platelet transmembrane potential detection method, which comprises: taking a washed platelet sample, carrying out re-suspending dilution with Hepes-Tyrodes buffer containing calcium chloride, adding to a flow tube, and recording as F; taking a washed platelet sample, carrying out re-suspending dilution with Hepes-Tyrodes buffer having 120-150 mmol/L of potassium chloride and 2-20 mmol/L of sodium chloride, adding to a flow tube, and recording as F0; adding gramicidin with a final concentration of 0.1-5 [mu]g/L to the F0 flow tube; adding DiBAC4(3) with a final concentration of 10-200 nmol/L to the F flow tube and the F0 flow tube, and dyeing; loading onto the machine and detecting; and calculating the result according to the corrected Nernst equation. According to the present invention, the method has characteristics of less platelet destruction, high sensitivity, good reproducibility, enrichment of the existing platelet storage quality assessment method, and prediction of the platelet storage damage.
Description
Technical field
The present invention relates to blood transfusion medical domain, specifically, relate to a kind of blood platelet membrane potential detection method.
Background technology
Blood platelet is one of blood constituent, plays an important role in hemostasis, blood coagulation and host immune.Blood platelet does not have nucleus, diameter about 2 ~ 3 μm.In normal blood, hematoblastic quantity is approximately 150 ~ 400 × 10
9/ L, MaLS is in vivo about 8 ~ 10 days.
Along with the development of blood transfusion medical science, platelet product infusion has become the important means of clinical blood transfusion treatment, for preventing and alleviating thrombopenia or dysfunction.But the Inefficacy of Platelets Transfusion that causes of Chang Fashengyin nonimmunologic factor clinically, the quality etc. as heating, infection, medicine, platelet product all may affect platelet transfusion effect.There is close contacting in the result for the treatment of of hematoblastic form, activation, metabolism and apoptosis and platelet transfusion.Blood platelet is subject to various factors in the process of collection, storage, infusion stimulates the storage damage produced, and causes the change of blood platelet structure and fuction.Hematoblastic storage may cause blood platelet part to be activated and the forfeiture of metabolic function, affects hematoblastic aggregation activity and adhesive capacity.The activation damage of blood platelet in vitro between storage life and Storage lesion of platelet decide the quality of platelet product.Correct rapid evaluation Storage lesion of platelet is for instructing Blood Transfusion Services and clinical treatment mechanism standard operation, evade platelet product and store damage, reduce the occurrence frequency of Inefficacy of Platelets Transfusion, improve blood transfusion curative effect, it is very necessary for saving limited blood resource.
The coherent detection index that current evaluate platelet preserves quality has platelet count, average external volume, morphology integration, pH, platelet aggregation activity, hypotonic shock rate, CD62p and phosphatidyl serine (PS) to express, the main change detecting platelet morphology and platelet function.There is not yet the detection that blood platelet organism electric energy is changed., namely there is the stable state of an environment in the various composition of intracellular environment and physicochemical property only change in very little scope under normal physiological conditions., just there is various pathophysiological change on this basis in intracellular environment homeostasis under the spread effect of inside and outside environmental factor.Blood platelet does not have nucleus, and thus extremely fragile, in vitro is very easy to destroyed.Because many factors is easily activated in vitro in blood platelet preservation process in vitro, the unbalance of blood platelet environment biological power stable state may be caused in this process, show as the change of blood platelet membrane potential.Thus the change of membrane potential is the reaction of initial, the first step of platelet activation damage.The very first time observes the unbalance of hematoblastic biological power, the i.e. measurement of blood platelet membrane potential, blood platelet physiological change can be understood rapidly more in time than additive method, the Storage lesion of platelet that anticipation is early possible, allocate blood resource as early as possible, reduce the generation of Inefficacy of Platelets Transfusion as far as possible, improve blood platelet clinical infusion safety.The monitoring of platelet membrane potential change can be used as platelet PLA2 conventional at present and function monitoring method one and supplements.
Current film potential detection method is as follows:
1. voltage clamp (Vol tage-clamp) is applied at first by British scholar Huxley and Katz.Its essence is by negative feedback micro current amplifier impressed current on excitable cells film, keep cellular transmembrane potentials constant, and control its numerical value rapidly, to observe the situation of membrance current under different film potential condition.The change of membrance current reflects the change of film resistance and membrane capacitance, and therefore voltage clamp can be used to the activity studying all ion channels on whole cell membrane or a bulk of cell membrane.But this technology owing to inserting two electrodes in cell, very large to cellular damage, be difficult to realize in cellule, again because cellular morphology is complicated, be difficult to keep the consistent of cell membrane biological nature everywhere, and gradually replace by patch-clamp.
2. patch clamp technique (Patch-clamp) is a kind of new technology that grows up on voltage clamp basis, has be clamp down on the method for film potential different at two: one from the key distinction of voltage clamp; Two is that the cell membrane area that current potential is fixed is different, and namely studied ion channel number is different.The same with voltage clamp, patch-clamp is also utilize negative feedback electronic circuit, and the cell membrane potential to several square micron adsorbed at microelectrode tip is fixed on certain level, observes the gas current flowing through passage.Its key realizing film potential fixing forms high resistance seals between glass microelectrode pointed edge and cell membrane, electrode tip opening part and the cell membrane zonule (diaphragm) connected are formed be from machinery or all very closely sealing-in in electricity, thus the molecule activity of single on cell (or most) ion channel can be reflected.
3. voltage sensitive dye is also referred to as fluorescence probe, can be divided into long response time voltage sensitive dye and fast response voltage sensitive dye according to dyestuff to the reaction velocity that film potential changes.Long response time dyestuff is called as Nernstian dyestuff, and cell membrane has permeability, and when cell is colored, dyestuff can infiltrate the two lipid layer of cell membrane.When cell membrane hyperpolarization, dyestuff combines with film component, and film surface dye concentration declines, and occur extinction effect, film surface fluorescence weakens; Otherwise when membrane depolarization, dyestuff is released into film surface again, film surface dye concentration increases, and film surface fluorescence strengthens.Under the impact of cell membrane both sides potential difference (PD), these voltage-sensitive fluorescent dyes obey Nernst equation in the outer distribution between dye solution and tenuigenin of born of the same parents, can calculate the size of cell membrane both sides potential difference (PD) and membrane potential accordingly.
The comparatively classical method of current mensuration cellular transmembrane potentials is microelectrode or patch clamp technique, but need the technical equipment of specialty and higher technical requirement with patch clamp measurements small hematoblastic membrane potential like this and waste time and energy, cost is higher, is unfavorable for making blood platelet membrane potential and change thereof detecting fast.Utilize compared with the methods such as the membrane potential Optical Mapping of voltage sensitive dye and traditional microelectrode, there is kinetic measurement conductivity, by the advantage that large electric jamming etc. is unique, receive extensive concern.But when detecting film potential with voltage sensitive dye, what spectrophotometer detected is total fluorescence intensity, may be subject to the impact of total cellular score, and can only relative quantification, and detection sensitivity is not high.In addition, using the monitoring of platelet membrane potential change as platelet PLA2 and function monitoring method, then require for platelet destruction less, possess good experimental repeatability, the Storage lesion of platelet that accurate anticipation is possible can be reached, improve the object of blood platelet clinical infusion safety.
In sum, high, few to platelet destruction and the blood platelet membrane potential detection method that experimental repeatability is good of a kind of detection sensitivity is needed badly.But have not been reported about these class methods at present.
Summary of the invention
The object of the invention is for deficiency of the prior art, a kind of blood platelet membrane potential detection method is provided.
For achieving the above object, the technical scheme that the present invention takes is:
A kind of blood platelet membrane potential detection method, described method comprises the following steps:
A) blood platelet bulk samples is centrifugal, abandons supernatant, washing;
B) platelet sample that part a) processes through step is got, the Hepes-Tyrodes buffer Eddy diffusion blood platelet that to use containing 0.8-5mmol/L lime chloride and KCE content be 1-6mmol/L, diluted blood platelet to 5 ~ 10 × 10
9/ L, adds streaming pipe, is designated as F; Separately get the platelet sample that part a) processes through step, use the Hepes-Tyrodes buffer Eddy diffusion blood platelet that KCE content is 120-150mmol/L, diluted blood platelet to 5 ~ 10 × 10
9/ L, adds streaming pipe, is designated as F
0;
C) to F
0add the short bacterium bar peptide solution that final concentration is 0.1-5 μ g/L in streaming pipe, make the complete depolarization of cell;
D) to F and F
0it is 10-200nmol/L DiBAC4 (3) solution that streaming pipe adds final concentration, shakes gently, mixing, darkroom room temperature dyeing 15-60min;
E) machine testing in streaming;
According to revised Nernst equation: V=E
dye=-(RT/ZF) * ln (F/F
0), calculate blood platelet membrane potential, wherein, F
0represent fluorescent value during the complete depolarization of cell, F represent cell be in quiescent condition under fluorescent value.
Preferably, steps d) in DiBAC4 (3) final concentration be 80-100nmol/L, step c) short-and-medium bacterium bar peptide final concentration is 0.5-2 μ g/L.
More preferably, steps d) in DiBAC4 (3) final concentration be 100nmol/L, step c) short-and-medium bacterium bar peptide final concentration is 1 μ g/L.
Preferably, steps d) in dyeing time be 15-22min.
More preferably, steps d) in dyeing time be 20min.
Preferably, step b) in, being designated as KCE content in the platelet sample Eddy diffusion of F Hepes-Tyrodesbuffer used is 1-5mmol/L, is designated as F
0platelet sample Eddy diffusion Hepes-Tyrodes buffer used in KCE content be 120-140mmol/L.
More preferably, step b) in, being designated as KCE content in the platelet sample Eddy diffusion of F Hepes-Tyrodesbuffer used is 2.7mmol/L, is designated as F
0platelet sample Eddy diffusion Hepes-Tyrodes buffer used in KCE content be 137mmol/L.
Preferably, step b) in, being designated as calcium chloride content in the platelet sample Eddy diffusion of F Hepes-Tyrodesbuffer used is 0.8-3mmol/L.
More preferably, step b) in, being designated as calcium chloride content in the platelet sample Eddy diffusion of F Hepes-Tyrodesbuffer used is 1.8mmol/L.
As a kind of embodiment of the present invention, described KCE content is the reagent of Hepes-Tyrodes buffer specifically containing following concentration of 1-6mmol/L: 137mmol/L sodium chloride, 2.7mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, pH are 7.4.
It should be noted that, the damping fluid that Hepes-Tyrodes buffer is well known to those skilled in the art, those skilled in the art should know, and the concentration of its each composition can have trickle change.
The use of flow cytometer is known to those skilled in the art, and as a kind of embodiment of the present invention, flow cytomery parameter is: voltage is arranged: FSC:E-1; SSC:313; FL1:555; FL2:150; FL3:150.But be not limited only to this, those skilled in the art reasonably can adjust according to actual conditions.
Revised Nernst equation: V=E
dye=-(RT/ZF) * ln (F/F
0) in, R represents universal gas constant, and T represents absolute temperature, Z represent dyestuff with electric charge.
As used herein, " room temperature " refers to temperature range well known in the art, is preferably 20-30 DEG C.
The invention has the advantages that:
1, the present invention observes the change of blood platelet membrane potential between storage life, for platelet quality monitoring provides a different angle and thinking, supplement as methodological, enrich morphology conventional at present and function assessment method carrys out evaluate platelet storage in vitro quality, the unbalance of hematoblastic biological power is observed by the very first time, understand blood platelet physiological change rapidly more in time, the Storage lesion of platelet that anticipation is early possible, allocate blood resource as early as possible, reduce the generation of Inefficacy of Platelets Transfusion as far as possible, improve blood platelet clinical infusion safety.
2, present invention incorporates low cytometric analysis, the average fluorescent strength of some cells can be obtained, and be the value of absolute fluorescence intensity, membrane potential mV numerical value directly can be obtained by calculating, and the membrane potential information of different conditions cell in cell can also be obtained, detection sensitivity is high.
3, present invention employs suitable dye well blood platelet depolarization reagent, in testing process, cytotoxicity is little, and platelet destruction is few, and detection sensitivity is high, and result is reproducible.
Embodiment
Below embodiment provided by the invention is elaborated.
The present invention uses voltage sensitive dye DiBAC4 (3) to detect blood platelet membrane potential.DiBAC4 (3) is monovalence negative ion long response time dyestuff, and unstressed configuration own, just sends fluorescence after entering cell and intracytoplasmic protein bound.During measurement, cell is immersed in fluorescent dye.Cell depolarization is represented when intensity of cellular fluorescence increase and membrane potential increase; Otherwise intensity of cellular fluorescence reduction and membrane potential reduce expression cell hyperpolarization.Nernst equation: V=E is obeyed in the distribution of DiBAC4 (3) in plasma membrane both sides
dye=-(RT/ZF) ln (C
i/ C
e), wherein R represents universal gas constant, and T represents absolute temperature, Z represent dyestuff with electric charge, C
i, C
erepresent the concentration of born of the same parents Inner, the outer free dye of born of the same parents respectively.Use flow cytomery F out represent full cell or organelle send by DiBAC4 (3) dyeing fluorescence.During cell complete depolarization, in born of the same parents, the outer fluorescent dye concentration of born of the same parents is identical, i.e. C
i=C
e, C
i/ C
ediBAC4 (3) fluorescence intensity F when the concentration of the outer free dye of born of the same parents can use blood platelet complete depolarization in born of the same parents
0replace F with fluorescence intensity under quiescent condition, Nernst equation now can be modified to: V=E
dye=-(RT/ZF) ln (F/F
0).
Embodiment 1 blood platelet membrane potential of the present invention detection method (one)
1 experiment material and instrument
Gather fresh blood platelet on the same day, put into blood platelet storage bag, 22 DEG C of concussions are preserved.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (purchased from Sigma) is dissolved in the working fluid that dimethyl sulfoxide (DMSO) is configured to 10 μm of ol/L ,-20 DEG C of preservations.
Short bacterium bar peptide (Gramicidin, purchased from Sigma, article No. 50845) use dimethyl sulfoxide (DMSO) to be configured to the working fluid of 2mg/mL ,-20 DEG C of preservations.
Hepes-Tyrodes buffer (without calcium): 137mmol/L sodium chloride, 2.7mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium): 2.7mmol/L sodium chloride, 137mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur); Hydro-extractor (Eppendorf, 5154D).
2 experimental procedures
1) get 1000 μ L Single-donor platelets stostes, be divided into 2 parts;
2) 800g, 5min are centrifugal, abandon supernatant, and PBS washs three times;
3) use 500 μ L Hepes-Tyrodes buffer (1.8mmol/L lime chloride) and Hepes-Tyrodesbuffer (high potassium) Eddy diffusion blood platelet respectively, diluted blood platelet is to about 5 ~ 10 × 10
9/ L, respectively flag F and F
0;
4) to F
0add the short bacterium bar peptide solution that final concentration is 1 μ g/L in streaming pipe, make the complete depolarization of cell; 5) to F and F
0it is 100nmol/L DiBAC4 (3) solution (DMSO concentration is no more than 0.1%) that streaming pipe adds final concentration, shakes gently, mixing; Darkroom room temperature dyeing 20min;
6) machine testing in streaming.Voltage is arranged: FSC:E-1; SSC:313; FL1:555; FL2:150; FL3:150.
According to revised Nernst equation: V=E
dye=-(RT/ZF) * ln (F/F
0), F
0represent fluorescent value during the complete depolarization of cell, F represent cell be in quiescent condition under fluorescent value, calculate blood platelet membrane potential.
3 experimental results
It is-56 ± 6mV that detection obtains 15 person-portion platelet membrane current potential mean values, similar with the result (-60mV) of Maclntyre gained to the result (-52 ± 8mV) of the patch-clamp method gained such as Lawrence T., illustrate that method of the present invention measures the accuracy of platelet membrane current potential very high.
Embodiment 2 blood platelet membrane potential of the present invention detection method (two)
1 experiment material and instrument
Gather fresh blood platelet on the same day, put into blood platelet storage bag, 22 DEG C of concussions are preserved.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (purchased from Sigma) is dissolved in the working fluid that dimethyl sulfoxide (DMSO) is configured to 10 μm of ol/L ,-20 DEG C of preservations.
Short bacterium bar peptide (Gramicidin, purchased from Sigma, article No. 50845) use dimethyl sulfoxide (DMSO) to be configured to the working fluid of 2mg/mL ,-20 DEG C of preservations.
Hepes-Tyrodes buffer (without calcium): 137mmol/L sodium chloride, 1mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium): 2mmol/L sodium chloride, 137mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur); Hydro-extractor (Eppendorf, 5154D).
2 experimental procedures
1) get 1000 μ L Single-donor platelets stostes, be divided into 2 parts;
2) 800g, 5min are centrifugal, abandon supernatant, and PBS washs three times;
3) use 500 μ L Hepes-Tyrodes buffer (0.8mmol/L lime chloride) and Hepes-Tyrodesbuffer (high potassium) Eddy diffusion blood platelet respectively, diluted blood platelet is to about 5 ~ 10 × 10
9/ L, respectively flag F and F
0;
4) to F
0add the short bacterium bar peptide solution that final concentration is 2 μ g/L in streaming pipe, make the complete depolarization of cell; 5) to F and F
0it is 80nmol/L DiBAC4 (3) solution (DMSO concentration is no more than 0.1%) that streaming pipe adds final concentration, shakes gently, mixing; Darkroom room temperature dyeing 15min;
6) machine testing in streaming.Voltage is arranged: FSC:E-1; SSC:313; FL1:555; FL2:150; FL3:150.
According to revised Nernst equation: V=E
dye=-(RT/ZF) * ln (F/F
0), F
0represent fluorescent value during the complete depolarization of cell, F represent cell be in quiescent condition under fluorescent value, calculate blood platelet membrane potential.
3 experimental results
It is-57.2 ± 6mV that detection obtains 15 person-portion platelet membrane current potential mean values, the result (-52 ± 8mV) done to the patch-clamp method such as Lawrence T. is similar with the result (-60mV) that Maclntyre does, and illustrates that method of the present invention measures the accuracy of platelet membrane current potential very high.
Embodiment 3 blood platelet membrane potential of the present invention detection method (three)
1 experiment material and instrument
Gather fresh blood platelet on the same day, put into blood platelet storage bag, 22 DEG C of concussions are preserved.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (purchased from Sigma) is dissolved in the working fluid that dimethyl sulfoxide (DMSO) is configured to 10 μm of ol/L ,-20 DEG C of preservations.
Short bacterium bar peptide (Gramicidin, purchased from Sigma, article No. 50845) use dimethyl sulfoxide (DMSO) to be configured to the working fluid of 2mg/mL ,-20 DEG C of preservations.
Hepes-Tyrodes buffer (without calcium): 134mmol/L sodium chloride, 5mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium): 17mmol/L sodium chloride, 120mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur); Hydro-extractor (Eppendorf, 5154D).
2 experimental procedures
1) get 1000 μ L Single-donor platelets stostes, be divided into 2 parts;
2) 800g, 5min are centrifugal, abandon supernatant, and PBS washs three times;
3) use 500 μ L Hepes-Tyrodes buffer (3mmol/L lime chloride) and Hepes-Tyrodesbuffer (high potassium) Eddy diffusion blood platelet respectively, diluted blood platelet is to about 5 ~ 10 × 10
9/ L, respectively flag F and F
0;
4) to F
0add the short bacterium bar peptide solution that final concentration is 0.5 μ g/L in streaming pipe, make the complete depolarization of cell;
5) to F and F
0it is 100nmol/L DiBAC4 (3) solution (DMSO concentration is no more than 0.1%) that streaming pipe adds final concentration, shakes gently, mixing; Darkroom room temperature dyeing 22min;
6) machine testing in streaming.Voltage is arranged: FSC:E-1; SSC:313; FL1:555; FL2:150; FL3:150.
According to revised Nernst equation: V=E
dye=-(RT/ZF) * ln (F/F
0), F
0represent fluorescent value during the complete depolarization of cell, F represent cell be in quiescent condition under fluorescent value, calculate blood platelet membrane potential.
3 experimental results
It is-55.8 ± 7mV that detection obtains 15 person-portion platelet membrane current potential mean values, the result (-52 ± 8mV) done to the patch-clamp method such as Lawrence T. is similar with the result (-60mV) that Maclntyre does, and illustrates that method of the present invention measures the accuracy of platelet membrane current potential very high.
Embodiment 4 blood platelet membrane potential of the present invention detection method (four)
1 experiment material and instrument
Gather fresh blood platelet on the same day, put into blood platelet storage bag, 22 DEG C of concussions are preserved.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (purchased from Sigma) is dissolved in the working fluid that dimethyl sulfoxide (DMSO) is configured to 10 μm of ol/L ,-20 DEG C of preservations.
Short bacterium bar peptide (Gramicidin, purchased from Sigma, article No. 50845) use dimethyl sulfoxide (DMSO) to be configured to the working fluid of 2mg/mL ,-20 DEG C of preservations.
Hepes-Tyrodes buffer (without calcium): 133mmol/L sodium chloride, 6mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium): 5mmol/L sodium chloride, 120mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur); Hydro-extractor (Eppendorf, 5154D).
2 experimental procedures
1) get 1000 μ L Single-donor platelets stostes, be divided into 2 parts;
2) 800g, 5min are centrifugal, abandon supernatant, and PBS washs three times;
3) use 500 μ L Hepes-Tyrodes buffer (5mmol/L lime chloride) and Hepes-Tyrodesbuffer (high potassium) Eddy diffusion blood platelet respectively, diluted blood platelet is to about 5 ~ 10 × 10
9/ L, respectively flag F and F
0;
4) to F
0add the short bacterium bar peptide solution that final concentration is 0.1 μ g/L in streaming pipe, make the complete depolarization of cell; 5) to F and F
0it is 200nmol/L DiBAC4 (3) solution (DMSO concentration is no more than 0.1%) that streaming pipe adds final concentration, shakes gently, mixing; Darkroom room temperature dyeing 15min;
6) machine testing in streaming.Voltage is arranged: FSC:E-1; SSC:313; FL1:555; FL2:150; FL3:150.
According to revised Nernst equation: V=E
dye=-(RT/ZF) * ln (F/F
0), F
0represent fluorescent value during the complete depolarization of cell, F represent cell be in quiescent condition under fluorescent value, calculate blood platelet membrane potential.
3 experimental results
It is-56.9 ± 6mV that detection obtains 15 person-portion platelet membrane current potential mean values, the result (-52 ± 8mV) done to the patch-clamp method such as Lawrence T. is similar with the result (-60mV) that Maclntyre does, and illustrates that method of the present invention measures the accuracy of platelet membrane current potential very high.
Embodiment 5 blood platelet membrane potential of the present invention detection method (five)
1 experiment material and instrument
Gather fresh blood platelet on the same day, put into blood platelet storage bag, 22 DEG C of concussions are preserved.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (purchased from Sigma) is dissolved in the working fluid that dimethyl sulfoxide (DMSO) is configured to 10 μm of ol/L ,-20 DEG C of preservations.
Short bacterium bar peptide (Gramicidin, purchased from Sigma, article No. 50845) use dimethyl sulfoxide (DMSO) to be configured to the working fluid of 2mg/mL ,-20 DEG C of preservations.
Hepes-Tyrodes buffer (without calcium): 137mmol/L sodium chloride, 1mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium): 8mmol/L sodium chloride, 130mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur); Hydro-extractor (Eppendorf, 5154D).
2 experimental procedures
1) get 1000 μ L Single-donor platelets stostes, be divided into 2 parts;
2) 800g, 5min are centrifugal, abandon supernatant, and PBS washs three times;
3) use 500 μ L Hepes-Tyrodes buffer (0.8mmol/L lime chloride) and Hepes-Tyrodesbuffer (high potassium) Eddy diffusion blood platelet respectively, diluted blood platelet is to about 5 ~ 10 × 10
9/ L, respectively flag F and F
0;
4) to F
0add the short bacterium bar peptide solution that final concentration is 5 μ g/L in streaming pipe, make the complete depolarization of cell; 5) to F and F
0it is 10nmol/L DiBAC4 (3) solution (DMSO concentration is no more than 0.1%) that streaming pipe adds final concentration, shakes gently, mixing; Darkroom room temperature dyeing 60min;
6) machine testing in streaming.Voltage is arranged: FSC:E-1; SSC:313; FL1:555; FL2:150; FL3:150.
According to revised Nernst equation: V=E
dye=-(RT/ZF) * ln (F/F
0), F
0represent fluorescent value during the complete depolarization of cell, F represent cell be in quiescent condition under fluorescent value, calculate blood platelet membrane potential.
3 experimental results
It is-55.9 ± 7mV that detection obtains 15 person-portion platelet membrane current potential mean values, the result (-52 ± 8mV) done to the patch-clamp method such as Lawrence T. is similar with the result (-60mV) that Maclntyre does, and illustrates that method of the present invention measures the accuracy of platelet membrane current potential very high.
The methodological study of embodiment 6 blood platelet membrane potential of the present invention detection method
The repeatability of the blood platelet membrane potential detection method of inspection embodiment 1-5.Concrete grammar is: the blood platelet membrane potential detecting 3 different people according to embodiment 1-5 either method, each detection 5 times, calculates mean value and the variance of blood platelet membrane potential.The Repeatability checking of embodiment 1 the results are shown in Table 1, and the Repeatability checking of embodiment 2-5 the results are shown in Table 2.Result shows blood platelet membrane potential detection method of the present invention repeatability very well.
The Repeatability checking result of table 1 embodiment 1
| Different person-portion | 1(mV) | 2(mV) | 3(mV) |
| 1 | -59.7 | -59.3 | -57.9 |
| 2 | -57.6 | -55.0 | -63.7 |
| 3 | -53.6 | -57.1 | -58.1 |
| 4 | -62.3 | -53.7 | -55.8 |
| 5 | -60.1 | -53.9 | -57.9 |
| Mean value | -58.66 | -55.8 | -58.68 |
| Variance | 3.28 | 2.37 | 2.96 |
The Repeatability checking result of table 2 embodiment 2-5
Comparative example 1
1 experiment material and instrument
Gather fresh blood platelet on the same day, put into blood platelet storage bag, 22 DEG C of concussions are preserved.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (purchased from Sigma) is dissolved in the working fluid that dimethyl sulfoxide (DMSO) is configured to 10 μm of ol/L ,-20 DEG C of preservations.
Hepes-Tyrodes buffer (without calcium): 137mmol/L sodium chloride, 2.7mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Blood platelet immobile liquid: use PBS to prepare 4% paraformaldehyde and 2% glutaraldehyde.
Flow cytometer (BD, FACSCalibur); Hydro-extractor (Eppendorf, 5154D).
2 experimental procedures
1) get 500 μ L Single-donor platelets stostes, 800g, 5min are centrifugal; Abandon supernatant, PBS washs three times.0.01% glutaraldehyde+2% paraformaldehyde fixes 2 hours under 4 DEG C of conditions, and 800g, 5min are centrifugal, and Hepes-Tyrodes buffer (without calcium) rinses three times.Hepes-Tyrodes buffer (adding final concentration is 1.8mmol/L lime chloride) diluted blood platelet is to about 5 ~ 10 × 10
9/ L, is labeled as F
0;
2) get 500 μ L Single-donor platelets stostes, 800g, 5min are centrifugal; Abandon supernatant, PBS washs three times.Hepes-Tyrodes buffer (without calcium) rinses three times.Hepes-Tyrodes buffer (adding final concentration is 1.8mmol/L lime chloride) Eddy diffusion blood platelet; Diluted blood platelet is to about 5 ~ 10 × 10
9/ L, is labeled as F;
3) to F and F
0it is 100nmol/L DiBAC4 (3) solution (DMSO concentration is no more than 0.1%) that streaming pipe adds final concentration, shakes gently, mixing.Darkroom room temperature dyeing 20min; Machine testing in streaming, voltage is arranged: FSC:E-1; SSC:313; FL1:555; FL2:150; FL3:150.
Detect the blood platelet membrane potential of 3 different people according to the method described above, each detection 5 times, calculate mean value and the variance of blood platelet membrane potential, the repeatability of inspection the method.
3 experimental results
The Repeatability checking of glutaraldehyde and paraformaldehyde fixation the results are shown in Table 3.Result shows that the repeatability of the method is significantly worse than method of the present invention.
The Repeatability checking result of table 3 glutaraldehyde and paraformaldehyde fixation
| Different person-portion | 1(mV) | 2(mV) | 3(mV) |
| 1 | -45.4 | -38.2 | -52.1 |
| 2 | -42.6 | -61.1 | -58.0 |
| 3 | -64,5 | -48.1 | -27.6 |
| 4 | -35.5 | -29.7 | -38.7 |
| 5 | -58.0 | -55.6 | -44.3 |
| Mean value | -49.2 | -46.6 | -44.1 |
| Variance | 10.5 | 12.7 | 11.8 |
Comparative example 2
1 experiment material and instrument
Gather fresh blood platelet on the same day, put into blood platelet storage bag, 22 DEG C of concussions are preserved.
Bis-oxonol (purchased from Sigma) is dissolved in the working fluid that dimethyl sulfoxide (DMSO) is configured to 10 μm of ol/L ,-20 DEG C of preservations.
Short bacterium bar peptide (Gramicidin, purchased from Sigma, article No. 50845) use dimethyl sulfoxide (DMSO) to be configured to the working fluid of 2mg/mL ,-20 DEG C of preservations.
Hepes-Tyrodes buffer (without calcium): 137mmol/L sodium chloride, 2.7mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium): 2.7mmol/L sodium chloride, 137mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur); Hydro-extractor (Eppendorf, 5154D).
2 experimental procedures
1) get 1000 μ L Single-donor platelets stostes, be divided into 2 parts;
2) 800g, 5min are centrifugal, abandon supernatant, and PBS washs three times;
3) use 500 μ L Hepes-Tyrodes buffer (1.8mmol/L lime chloride) and Hepes-Tyrodesbuffer (high potassium) Eddy diffusion blood platelet respectively, diluted blood platelet is to about 5 ~ 10 × 10
9/ L, respectively flag F and F
0;
4) to F
0add the short bacterium bar peptide solution that final concentration is 1 μ g/L in streaming pipe, make the complete depolarization of cell;
5) to F and F
0it is 100nmol/L Bis-oxonol solution (DMSO concentration is no more than 0.1%) that streaming pipe adds final concentration, shakes gently, mixing; Darkroom room temperature dyeing 20min;
6) machine testing in streaming.Voltage is arranged: FSC:E-1; SSC:313; FL1:555; FL2:150; FL3:150.
According to revised Nernst equation: V=E
dye=-(RT/ZF) * ln (F/F
0), F
0represent fluorescent value during the complete depolarization of cell, F represent cell be in quiescent condition under fluorescent value, calculate blood platelet membrane potential.
Detect the blood platelet membrane potential of 3 different people according to the method described above, each detection 5 times, calculate mean value and the variance of blood platelet membrane potential, the repeatability of inspection the method.
3 experimental results
It is-45.8 ± 10mV that detection obtains 15 person-portion platelet membrane current potential mean values, the result (-60mV) that the result (-52 ± 8mV) done with the patch-clamp method such as Lawrence T. and Maclntyre do differs comparatively large, illustrates that the accuracy of the method mensuration platelet membrane current potential is general.
The Repeatability checking of the method the results are shown in Table 4.Result shows that the repeatability of the method is significantly worse than method of the present invention.
The Repeatability checking result of table 4 the present embodiment method
| Different person-portion | 1(mV) | 2(mV) | 3(mV) |
| Mean value | -43.8 | -41.7 | -49.2 |
| Variance | 9.8 | 10.7 | 10.4 |
Comparative example 3
1 experiment material and instrument
Gather fresh blood platelet on the same day, put into blood platelet storage bag, 22 DEG C of concussions are preserved.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (purchased from Sigma) is dissolved in the working fluid that dimethyl sulfoxide (DMSO) is configured to 10 μm of ol/L ,-20 DEG C of preservations.
Short bacterium bar peptide (Gramicidin, purchased from Sigma, article No. 50845) use dimethyl sulfoxide (DMSO) to be configured to the working fluid of 2mg/mL ,-20 DEG C of preservations.
Hepes-Tyrodes buffer (without calcium): 133mmol/L sodium chloride, 6mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium): 5mmol/L sodium chloride, 120mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L NaOH adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur); Hydro-extractor (Eppendorf, 5154D).
2 experimental procedures
7) get 1000 μ L Single-donor platelets stostes, be divided into 2 parts;
8) 800g, 5min are centrifugal, abandon supernatant, and PBS washs three times;
9) use 500 μ L Hepes-Tyrodes buffer (5mmol/L lime chloride) and Hepes-Tyrodesbuffer (high potassium) Eddy diffusion blood platelet respectively, diluted blood platelet is to about 5 ~ 10 × 10
9/ L, respectively flag F and F
0;
10) to F
0add the short bacterium bar peptide solution that final concentration is 0.1 μ g/L in streaming pipe, make the complete depolarization of cell;
11) to F and F
0it is 220nmol/L DiBAC4 (3) solution (DMSO concentration is no more than 0.1%) that streaming pipe adds final concentration, shakes gently, mixing; Darkroom room temperature dyeing 15min;
12) machine testing in streaming.Voltage is arranged: FSC:E-1; SSC:313; FL1:555; FL2:150; FL3:150.
According to revised Nernst equation: V=E
dye=-(RT/ZF) * ln (F/F
0), F
0represent fluorescent value during the complete depolarization of cell, F represent cell be in quiescent condition under fluorescent value, calculate blood platelet membrane potential.
Detect the blood platelet membrane potential of 3 different people according to the method described above, each detection 5 times, calculate mean value and the variance of blood platelet membrane potential, the repeatability of inspection the method.
3 experimental results
It is-53.6 ± 8mV that detection obtains 15 person-portion platelet membrane current potential mean values, similar with the result (-60mV) of Maclntyre gained to the result (-52 ± 8mV) of the patch-clamp method gained such as Lawrence T., illustrate that the method measures the accuracy of platelet membrane current potential higher.
The Repeatability checking of the method the results are shown in Table 5.Result shows that the repeatability of the method is significantly worse than method of the present invention.
The Repeatability checking result of table 5 the present embodiment method
| Different person-portion | 1(mV) | 2(mV) | 3(mV) |
| Mean value | -49.0 | -53.4 | -52.9 |
| Variance | 7.9 | 8.3 | 8.0 |
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (10)
1. a blood platelet membrane potential detection method, is characterized in that, comprises the following steps:
A) blood platelet bulk samples is centrifugal, abandons supernatant, washing;
B) platelet sample that part a) processes through step is got, the Hepes-Tyrodes buffer Eddy diffusion blood platelet that to use containing 0.8-5mmol/L lime chloride and KCE content be 1-6mmol/L, diluted blood platelet to 5 ~ 10 × 10
9/ L, adds streaming pipe, is designated as F; Separately get the platelet sample that part a) processes through step, use KCE content for 120-150mmol/L and sodium chloride content be 2-20mmol/L Hepes-Tyrodes buffer Eddy diffusion blood platelet, diluted blood platelet to 5 ~ 10 × 10
9/ L, adds streaming pipe, is designated as F
0;
C) to F
0add the short bacterium bar peptide solution that final concentration is 0.1-5 μ g/L in streaming pipe, make the complete depolarization of cell;
D) to F and F
0it is 10-200nmol/L DiBAC4 (3) solution that streaming pipe adds final concentration, shakes gently, mixing, darkroom room temperature dyeing 15-60min;
E) machine testing in streaming;
According to revised Nernst equation: V=E
dye=-(RT/ZF) * ln (F/F
0), calculate blood platelet membrane potential, wherein, F
0represent fluorescent value during the complete depolarization of cell, F represent cell be in quiescent condition under fluorescent value.
2. blood platelet membrane potential detection method according to claim 1, is characterized in that, steps d) in DiBAC4 (3) final concentration be 80-100nmol/L, step c) short-and-medium bacterium bar peptide final concentration is 0.5-2 μ g/L.
3. blood platelet membrane potential detection method according to claim 2, is characterized in that, steps d) in DiBAC4 (3) final concentration be 100nmol/L, step c) short-and-medium bacterium bar peptide final concentration is 1 μ g/L.
4. blood platelet membrane potential detection method according to claim 1, is characterized in that, steps d) in dyeing time be 15-22min.
5. blood platelet membrane potential detection method according to claim 4, is characterized in that, steps d) in dyeing time be 20min.
6. blood platelet according to claim 1 expands film potential detection method, it is characterized in that, step b) in, being designated as KCE content in the platelet sample Eddy diffusion of F Hepes-Tyrodes buffer used is 1-5mmol/L, is designated as F
0platelet sample Eddy diffusion Hepes-Tyrodes buffer used in KCE content be 120-140mmol/L.
7. blood platelet according to claim 6 expands film potential detection method, it is characterized in that, step b) in, being designated as KCE content in the platelet sample Eddy diffusion of F Hepes-Tyrodes buffer used is 2.7mmol/L, is designated as F
0platelet sample Eddy diffusion Hepes-Tyrodes buffer used in KCE content be 137mmol/L.
8. blood platelet according to claim 1 expands film potential detection method, it is characterized in that, step b) in, being designated as calcium chloride content in the platelet sample Eddy diffusion of F Hepes-Tyrodes buffer used is 0.8-3mmol/L.
9. blood platelet according to claim 8 expands film potential detection method, it is characterized in that, step b) in, being designated as calcium chloride content in the platelet sample Eddy diffusion of F Hepes-Tyrodes buffer used is 1.8mmol/L.
10. blood platelet membrane potential detection method according to claim 1, it is characterized in that, described KCE content is the reagent of Hepes-Tyrodes buffer specifically containing following concentration of 1-6mmol/L: 137mmol/L sodium chloride, 2.7mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 1mmol/L magnesium chloride, 0.4mmol/L sodium dihydrogen phosphate, 5.5mmol/L glucose, 10mmol/L Hepes, pH are 7.4.
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