CN104964909B - A kind of blood platelet membrane potential detection method - Google Patents
A kind of blood platelet membrane potential detection method Download PDFInfo
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- CN104964909B CN104964909B CN201510280527.9A CN201510280527A CN104964909B CN 104964909 B CN104964909 B CN 104964909B CN 201510280527 A CN201510280527 A CN 201510280527A CN 104964909 B CN104964909 B CN 104964909B
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Abstract
The present invention relates to a kind of blood platelet membrane potential detection method, step is：The platelet sample after washing is taken to be suspended again dilution with the Hepes Tyrodes buffer of chloride containing calcium, addition streaming pipe, is denoted as F；The another platelet sample potassium chloride that takes after washing be 120 150mmol/L, and the Hepes Tyrodes buffer that sodium chloride is 2 20mmol/L suspend dilution again, and addition streaming pipe, is denoted as F0；To F0The short bacterium bar peptide of final concentration of 0.1 5 μ g/L is added in streaming pipe；To F and F0Streaming pipe adds final concentration of 10 200nmol/L DiBAC4 (3), dyeing；Upper machine testing；According to revised Nernst equation result of calculation.This method platelet destruction is few, and high sensitivity is reproducible, has enriched existing blood platelet and has preserved method for evaluating quality, can prejudge Storage lesion of platelet.
Description
Technical field
The present invention relates to blood transfusion medical domain, specifically, it is related to a kind of blood platelet membrane potential detection method.
Background technology
Blood platelet is one of blood constituent, is played an important role in terms of hemostasis, blood coagulation and host immune.Blood platelet
There is no nucleus, about 2~3 μm of diameter.Hematoblastic quantity is about 150~400 × 10 in normal blood9/ L, in vivo
MaLS is about 8~10 days.
With the development of blood transfusion medicine, platelet product infusion has become the important means of clinical blood transfusion treatment, for pre-
Anti- and alleviation thrombopenia or dysfunction.But clinically platelet transfusion caused by Chang Fashengyin nonimmunologic factors without
Effect, such as fever, infection, medicine, the quality of platelet product may influence platelet transfusion effect.Hematoblastic form,
There is close contact in activation, metabolism and the therapeutic effect of apoptosis and platelet transfusion.Blood platelet is in collection, storage, infusion
During be subject to various factors stimulate produce storage damage, cause the change of blood platelet structure and function.Hematoblastic storage
Depositing may cause blood platelet part to be activated and the forfeiture of metabolic function, influence hematoblastic aggregation activity and adhesive capacity.
Activation during blood platelet preserves in vitro damages the quality that i.e. Storage lesion of platelet decides platelet product.It is correct quick
Assessment Storage lesion of platelet evades platelet product storage for instructing Blood Transfusion Services and clinical treatment mechanism standard operation
Damage, reduces the occurrence frequency of Inefficacy of Platelets Transfusion, improves blood transfusion curative effect, it is very must to save for limited blood resource
Want.
At present assessment blood platelet preserve quality coherent detection index have platelet count, average external volume, morphology integration,
PH, platelet aggregation activity, hypotonic shock rate, CD62p and phosphatidyl serine (PS) expression etc., predominantly detect platelet PLA2
And the change of platelet function.There is not yet the detection changed to blood platelet organism electric energy.It is intracellular under normal physiological conditions
The various composition and physicochemical property of environment only change in the range of very little, i.e. the stable state there are an interior environment.Inside,
, just there are various pathophysiological changes on this basis in intracellular environment homeostasis under the stimulation of external environment factor.Blood
Platelet does not have nucleus, thus extremely fragile, and in vitro is very easy to be destroyed.Blood platelet preserve in vitro during by
Easily it is activated in vitro in many factors, the unbalance of environment biological power stable state in blood platelet may be caused during this,
Show as the change of blood platelet membrane potential.Thus the change of membrane potential is initial, the first step of platelet activation damage
Reaction.The measurement of the unbalance of hematoblastic biological power, i.e. blood platelet membrane potential is observed at the first time, can be more than other methods
Blood platelet physiological change is understood quickly in time, prejudges possible Storage lesion of platelet early, allocates blood resource as early as possible, to the greatest extent
Amount reduces the generation of Inefficacy of Platelets Transfusion, improves blood platelet clinical infusion safety.The monitoring of platelet membrane potential change can be made
For currently used platelet PLA2 and a supplement of function monitoring method.
Film potential detection method is as follows at present：
1. voltage clamp (Vol tage-clamp) is applied at first by British scholar Huxley and Katz.Its essence is logical
Negative-feedback micro current amplifier impressed current on excitable cells film is crossed, keeps cellular transmembrane potentials constant, and control rapidly
Its numerical value, to observe the situation of the membrance current under the conditions of different film potentials.The change of membrance current reflects film resistance and membrane capacitance
Change, therefore voltage clamp can be used to study the activity of all ion channels on whole cell membrane or a bulk of cell membrane.But should
Technology is very big to cellular damage due to being inserted into two electrodes in the cell, is difficult to realize in cellule, and because cellular morphology is answered
It is miscellaneous, it is difficult to keep the consistent of cell membrane biological nature everywhere, and gradually substituted by patch-clamp.
2. patch clamp technique (Patch-clamp) is a kind of new technology that grows up on the basis of voltage clamp, with voltage clamp
The main distinction have two：First, the method for clamping down on film potential is different；Second, the cell membrane area that current potential is fixed is different, that is, studied
Ion channel number it is different.As voltage clamp, patch-clamp is inhaled microelectrode tip also with negative-feedback electronic circuit
The cell membrane potential of attached one to several square microns is fixed on certain level, and the gas current of passage is flowed through in observation.In fact
The key that existing film potential is fixed is to form high resistance seals between glass microelectrode pointed edge and cell membrane, makes electrode tip opening
Locate to form either still electrically all extremely tight sealing-ins from machinery with the cell membrane zonule (diaphragm) to connect, so that
It can reflect the molecule activity of (or most) ion channel single on cell.
3. voltage sensitive dye is also referred to as fluorescence probe, the reaction speed that film potential changes can be divided into slowly according to dyestuff
Response voltage sensitive dye and fast response voltage sensitive dye.Long response time dyestuff is referred to as Nernstian dyestuffs, has to cell membrane
Permeability, when cell is colored, dyestuff can penetrate into the double lipid layers of cell membrane.When cell membrane hyperpolarization, dyestuff and film component phase
With reference to film surface dye strength declines, and extinction effect, film surface fluorescent weakening occurs；Conversely, when membrane depolarization, dye
Material is released into film surface, the increase of film surface dye strength, film surface Fluorescence Increasing again.The potential difference in cell membrane both sides
Under the influence of, Nernst equation is obeyed in distribution of these voltage-sensitive fluorescent dyes between extracellular dye solution and cytoplasm,
The cell membrane both sides potential difference i.e. size of membrane potential can be calculated accordingly.
The more classical method of measure cellular transmembrane potentials is microelectrode or patch clamp technique at present, but uses patch-clamp
The so small hematoblastic membrane potential of measurement needs the technical equipment and higher technical requirements and time-consuming and laborious, cost of specialty
It is higher, it is unfavorable for making quick detection to blood platelet membrane potential and its change.Utilize the membrane potential of voltage sensitive dye
Optical Mapping has dynamic measurement electrical conduction, from uniquenesses such as big electric jammings compared with the methods of traditional microelectrode
Advantage, have received widespread attention.However, when detecting film potential with voltage sensitive dye, spectrophotometer detection is total
Fluorescence intensity, may be influenced be subject to total number of cells, and can only relative quantification, detection sensitivity is high.In addition, by blood platelet
Film potential change monitoring be used as platelet PLA2 and function monitoring method, then requirement it is less for platelet destruction, possess compared with
Good experimental repeatability, can reach and accurately prejudge possible Storage lesion of platelet, improve blood platelet clinical infusion safety
Purpose.
In conclusion there is an urgent need for a kind of detection sensitivity is high, few to platelet destruction and experimental repeatability is good blood platelet across
Film potential detection method.But had not been reported at present on this kind of method.
The content of the invention
The purpose of the present invention is for deficiency of the prior art, there is provided a kind of blood platelet membrane potential detection method.
To achieve the above object, the present invention adopts the technical scheme that：
A kind of blood platelet membrane potential detection method, the method comprise the following steps：
A) blood platelet bulk samples centrifuge, and abandon supernatant, wash；
B) platelet sample for taking part to be handled through step a), uses calcium chloride containing 0.8-5mmol/L and KCE content
Suspend again blood platelet for the Hepes-Tyrodes buffer of 1-6mmol/L, dilution blood platelet to 5~10 × 109/ L, adds
Streaming pipe, is denoted as F；The platelet sample that part is handled through step a) separately is taken, the use of KCE content is 120-150mmol/L's
Hepes-Tyrodes buffer suspend blood platelet again, dilution blood platelet to 5~10 × 109/ L, adds streaming pipe, is denoted as F0；
C) to F0The short bacterium bar peptide solution of final concentration of 0.1-5 μ g/L is added in streaming pipe, cell is depolarized completely；
D) to F and F0Streaming pipe adds final concentration of 10-200nmol/L DiBAC4 (3) solution, gently shakes, and mixes,
Darkroom room temperature dyeing 15-60min；
E) machine testing in streaming；
According to revised Nernst equation：V=Edye=-(RT/ZF) * ln (F/F0), blood platelet membrane potential is calculated,
Wherein, F0Fluorescent value when cell depolarizes completely is represented, F represents the fluorescent value that cell is under quiescent condition.
Preferably, short bacterium bar peptide is final concentration of in DiBAC4 (3) final concentration of 80-100nmol/L, step c) in step d)
0.5-2μg/L。
It is highly preferred that short bacterium bar peptide final concentration of 1 in DiBAC4 (3) final concentration of 100nmol/L, step c) in step d)
μg/L。
Preferably, dyeing time is 15-22min in step d).
It is highly preferred that dyeing time is 20min in step d).
Preferably, in step b), the platelet sample for being denoted as F suspends in Hepes-Tyrodes buffer used again
KCE content is 1-5mmol/L, is denoted as F0Platelet sample suspend again in Hepes-Tyrodes buffer used
KCE content is 120-140mmol/L.
It is highly preferred that in step b), the platelet sample for being denoted as F suspends Hepes-Tyrodes buffer used again
Middle KCE content is 2.7mmol/L, is denoted as F0Platelet sample suspend again Hepes-Tyrodes buffer used
Middle KCE content is 137mmol/L.
Preferably, in step b), the platelet sample for being denoted as F suspends in Hepes-Tyrodes buffer used again
Calcium chloride content is 0.8-3mmol/L.
It is highly preferred that in step b), the platelet sample for being denoted as F suspends Hepes-Tyrodes buffer used again
Middle calcium chloride content is 1.8mmol/L.
As a kind of embodiment of the present invention, the KCE content is the Hepes- of 1-6mmol/L
Tyrodes buffer specifically contain the reagent of following concentration：137mmol/L sodium chloride, 2.7mmol/L potassium chloride, 12mmol/L
Sodium acid carbonate, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, pH
For 7.4.
It should be noted that the buffer solution that Hepes-Tyrodes buffer are well known to those skilled in the art, ability
Field technique personnel will be appreciated that the concentration of its each component can have trickle change.
The use of flow cytometer is well known to those skilled in the art, as a kind of specific real of the present invention
Mode is applied, flow cytomery parameter is：Voltage is set：FSC：E-1；SSC：313；FL1：555；FL2：150；FL3：150.
But this is not limited only to, those skilled in the art can reasonably be adjusted according to actual conditions.
Revised Nernst equation：V=Edye=-(RT/ZF) * ln (F/F0) in, R represents universal gas constant, T generations
Table absolute temperature, Z represent the electric charge of dyestuff institute band.
As used herein, " room temperature " refers to temperature range well known in the art, preferably 20-30 DEG C.
The invention has the advantages that：
1st, change of the present invention to blood platelet membrane potential during preservation is observed, and is provided for platelet quality monitoring
One different angle and thinking, as the supplement of methodology, have enriched currently used morphology and function assessment method to comment
Estimate blood platelet storage in vitro quality, can be by observing the unbalance of hematoblastic biological power at the first time, more in time rapidly
Blood platelet physiological change is solved, possible Storage lesion of platelet is prejudged early, allocates blood resource as early as possible, reduce blood platelet to the greatest extent
Invalid generation is transfused, improves blood platelet clinical infusion safety.
2nd, present invention incorporates low cytometric analysis, the average fluorescent strength of certain amount cell can be obtained, and is
The value of absolute fluorescence intensity, membrane potential mV numerical value can be directly obtained by calculating, and can also obtain different shapes in cell
The membrane potential information of state cell, detection sensitivity are high.
3rd, present invention employs suitable dyestuff and blood platelet to depolarize reagent, and cytotoxicity is small in detection process, and blood is small
Plate destroys few, detection sensitivity height, as a result reproducible.
Embodiment
Elaborate below to embodiment provided by the invention.
The present invention detects blood platelet membrane potential using voltage sensitive dye DiBAC4 (3).DiBAC4 (3) bears for monovalence
Ion long response time dyestuff, unstressed configuration itself, fluorescence is just sent after being combined into cell with intracytoplasmic protein.It is thin during measurement
Born of the same parents are immersed in fluorescent dye.When intensity of cellular fluorescence increase is that membrane potential increase represents cell depolarization；It is conversely, intracellular
It is that membrane potential reduces expression cell hyperpolarization that fluorescence intensity, which reduces,.Distributions of the DiBAC4 (3) in plasma membrane both sides is obeyed can this spy
Equation：V=Edye=-(RT/ZF) ln (Ci/Ce), wherein R represents universal gas constant, and T represents absolute temperature, and Z represents dyestuff institute
The electric charge of band, Ci、CeBorn of the same parents Inner, the concentration of extracellular free dye are represented respectively.The F come out using flow cytomery represents complete
Cell or organelle send fluorescence by DiBAC4 (3) dyeing.Intracellular extracellular fluorescent dye concentration when cell depolarizes completely
It is identical, i.e. Ci=Ce, Ci/CeDiBAC4 (3) fluorescence when blood platelet can be used to depolarize completely for the concentration of the extracellular free dye of intracellular
Intensity F0Substitute F with fluorescence intensity under quiescent condition, Nernst equation at this time can be modified to：V=Edye=-(RT/ZF) ln
(F/F0)。
The blood platelet membrane potential detection method (one) of the present invention of embodiment 1
1 experiment material and instrument
Fresh blood platelet, is put into blood platelet storage bag on the day of collection, and 22 DEG C of concussions preserve.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (being purchased from Sigma)
It is dissolved in the working solution 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) is configured to 2mg/mL's using dimethyl sulfoxide (DMSO)
Working solution, -20 DEG C of preservations.
Hepes-Tyrodes buffer (no calcium)：137mmol/L sodium chloride, 2.7mmol/L potassium chloride, 12mmol/L carbon
Sour hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/
L sodium hydroxides adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium)：2.7mmol/L sodium chloride, 137mmol/L potassium chloride, 12mmol/L carbon
Sour hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/
L sodium hydroxides adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur)；Centrifuge (Eppendorf, 5154D).
2 experimental procedures
1) 1000 μ L Single-donor platelets stostes are taken, are divided into 2 parts；
2) 800g, 5min are centrifuged, and abandon supernatant, and PBS is washed three times；
3) 500 μ L Hepes-Tyrodes buffer (1.8mmol/L calcium chloride) and Hepes-Tyrodes are used respectively
Again suspend buffer (high potassium) blood platelet, dilution blood platelet to about 5~10 × 109/ L, difference flag F and F0；
4) to F0The short bacterium bar peptide solution of final concentration of 1 μ g/L is added in streaming pipe, cell is depolarized completely；5) to F
And F0Streaming pipe adds final concentration of 100nmol/L DiBAC4 (3) solution (DMSO concentration is no more than 0.1%), gently shakes,
Mix；Darkroom room temperature dyeing 20min；
6) machine testing in streaming.Voltage is set：FSC：E-1；SSC：313；FL1：555；FL2：150；FL3：150.
According to revised Nernst equation：V=Edye=-(RT/ZF) * ln (F/F0), F0Cell is represented to depolarize completely
When fluorescent value, F represents the fluorescent value that cell is under quiescent condition, calculates blood platelet membrane potential.
3 experimental results
To obtain 15 person-portion platelet membrane current potential average values be -56 ± 6mV for detection, with the patch-clamp method institute such as Lawrence T.
The result (- 60mV) obtained by result (- 52 ± 8mV) and Maclntyre obtained is similar, illustrates the method measure blood platelet of the present invention
The accuracy of film potential is very high.
The blood platelet membrane potential detection method (two) of the present invention of embodiment 2
1 experiment material and instrument
Fresh blood platelet, is put into blood platelet storage bag on the day of collection, and 22 DEG C of concussions preserve.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (being purchased from Sigma)
It is dissolved in the working solution 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) is configured to 2mg/mL's using dimethyl sulfoxide (DMSO)
Working solution, -20 DEG C of preservations.
Hepes-Tyrodes buffer (no calcium)：137mmol/L sodium chloride, 1mmol/L potassium chloride, 12mmol/L carbonic acid
Hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L
Sodium hydroxide adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium)：2mmol/L sodium chloride, 137mmol/L potassium chloride, 12mmol/L carbonic acid
Hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L
Sodium hydroxide adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur)；Centrifuge (Eppendorf, 5154D).
2 experimental procedures
1) 1000 μ L Single-donor platelets stostes are taken, are divided into 2 parts；
2) 800g, 5min are centrifuged, and abandon supernatant, and PBS is washed three times；
3) 500 μ L Hepes-Tyrodes buffer (0.8mmol/L calcium chloride) and Hepes-Tyrodes are used respectively
Again suspend buffer (high potassium) blood platelet, dilution blood platelet to about 5~10 × 109/ L, difference flag F and F0；
4) to F0The short bacterium bar peptide solution of final concentration of 2 μ g/L is added in streaming pipe, cell is depolarized completely；5) to F
And F0Streaming pipe adds final concentration of 80nmol/L DiBAC4 (3) solution (DMSO concentration is no more than 0.1%), gently shakes, mixes
It is even；Darkroom room temperature dyeing 15min；
6) machine testing in streaming.Voltage is set：FSC：E-1；SSC：313；FL1：555；FL2：150；FL3：150.
According to revised Nernst equation：V=Edye=-(RT/ZF) * ln (F/F0), F0Cell is represented to depolarize completely
When fluorescent value, F represents the fluorescent value that cell is under quiescent condition, calculates blood platelet membrane potential.
3 experimental results
To obtain 15 person-portion platelet membrane current potential average values be -57.2 ± 6mV for detection, with the patch-clamp method such as Lawrence T.
The result (- 60mV) that the result (- 52 ± 8mV) and Maclntyre done are done is similar, illustrates that the method measure blood of the present invention is small
The accuracy of plate film potential is very high.
The blood platelet membrane potential detection method (three) of the present invention of embodiment 3
1 experiment material and instrument
Fresh blood platelet, is put into blood platelet storage bag on the day of collection, and 22 DEG C of concussions preserve.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (being purchased from Sigma)
It is dissolved in the working solution 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) is configured to 2mg/mL's using dimethyl sulfoxide (DMSO)
Working solution, -20 DEG C of preservations.
Hepes-Tyrodes buffer (no calcium)：134mmol/L sodium chloride, 5mmol/L potassium chloride, 12mmol/L carbonic acid
Hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L
Sodium hydroxide adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium)：17mmol/L sodium chloride, 120mmol/L potassium chloride, 12mmol/L carbon
Sour hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/
L sodium hydroxides adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur)；Centrifuge (Eppendorf, 5154D).
2 experimental procedures
1) 1000 μ L Single-donor platelets stostes are taken, are divided into 2 parts；
2) 800g, 5min are centrifuged, and abandon supernatant, and PBS is washed three times；
3) 500 μ L Hepes-Tyrodes buffer (3mmol/L calcium chloride) and Hepes-Tyrodes are used respectively
Again suspend buffer (high potassium) blood platelet, dilution blood platelet to about 5~10 × 109/ L, difference flag F and F0；
4) to F0The short bacterium bar peptide solution of final concentration of 0.5 μ g/L is added in streaming pipe, cell is depolarized completely；
5) to F and F0Streaming pipe adds final concentration of 100nmol/L DiBAC4 (3) solution, and (DMSO concentration is no more than
0.1%), gently shake, mix；Darkroom room temperature dyeing 22min；
6) machine testing in streaming.Voltage is set：FSC：E-1；SSC：313；FL1：555；FL2：150；FL3：150.
According to revised Nernst equation：V=Edye=-(RT/ZF) * ln (F/F0), F0Cell is represented to depolarize completely
When fluorescent value, F represents the fluorescent value that cell is under quiescent condition, calculates blood platelet membrane potential.
3 experimental results
To obtain 15 person-portion platelet membrane current potential average values be -55.8 ± 7mV for detection, with the patch-clamp method such as Lawrence T.
The result (- 60mV) that the result (- 52 ± 8mV) and Maclntyre done are done is similar, illustrates that the method measure blood of the present invention is small
The accuracy of plate film potential is very high.
The blood platelet membrane potential detection method (four) of the present invention of embodiment 4
1 experiment material and instrument
Fresh blood platelet, is put into blood platelet storage bag on the day of collection, and 22 DEG C of concussions preserve.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (being purchased from Sigma)
It is dissolved in the working solution 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) is configured to 2mg/mL's using dimethyl sulfoxide (DMSO)
Working solution, -20 DEG C of preservations.
Hepes-Tyrodes buffer (no calcium)：133mmol/L sodium chloride, 6mmol/L potassium chloride, 12mmol/L carbonic acid
Hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L
Sodium hydroxide adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium)：5mmol/L sodium chloride, 120mmol/L potassium chloride, 12mmol/L carbonic acid
Hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L
Sodium hydroxide adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur)；Centrifuge (Eppendorf, 5154D).
2 experimental procedures
1) 1000 μ L Single-donor platelets stostes are taken, are divided into 2 parts；
2) 800g, 5min are centrifuged, and abandon supernatant, and PBS is washed three times；
3) 500 μ L Hepes-Tyrodes buffer (5mmol/L calcium chloride) and Hepes-Tyrodes are used respectively
Again suspend buffer (high potassium) blood platelet, dilution blood platelet to about 5~10 × 109/ L, difference flag F and F0；
4) to F0The short bacterium bar peptide solution of final concentration of 0.1 μ g/L is added in streaming pipe, cell is depolarized completely；5) to
F and F0Streaming pipe adds final concentration of 200nmol/L DiBAC4 (3) solution (DMSO concentration is no more than 0.1%), gently shakes,
Mix；Darkroom room temperature dyeing 15min；
6) machine testing in streaming.Voltage is set：FSC：E-1；SSC：313；FL1：555；FL2：150；FL3：150.
According to revised Nernst equation：V=Edye=-(RT/ZF) * ln (F/F0), F0Cell is represented to depolarize completely
When fluorescent value, F represents the fluorescent value that cell is under quiescent condition, calculates blood platelet membrane potential.
3 experimental results
To obtain 15 person-portion platelet membrane current potential average values be -56.9 ± 6mV for detection, with the patch-clamp method such as Lawrence T.
The result (- 60mV) that the result (- 52 ± 8mV) and Maclntyre done are done is similar, illustrates that the method measure blood of the present invention is small
The accuracy of plate film potential is very high.
The blood platelet membrane potential detection method (five) of the present invention of embodiment 5
1 experiment material and instrument
Fresh blood platelet, is put into blood platelet storage bag on the day of collection, and 22 DEG C of concussions preserve.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (being purchased from Sigma)
It is dissolved in the working solution 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) is configured to 2mg/mL's using dimethyl sulfoxide (DMSO)
Working solution, -20 DEG C of preservations.
Hepes-Tyrodes buffer (no calcium)：137mmol/L sodium chloride, 1mmol/L potassium chloride, 12mmol/L carbonic acid
Hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L
Sodium hydroxide adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium)：8mmol/L sodium chloride, 130mmol/L potassium chloride, 12mmol/L carbonic acid
Hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L
Sodium hydroxide adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur)；Centrifuge (Eppendorf, 5154D).
2 experimental procedures
1) 1000 μ L Single-donor platelets stostes are taken, are divided into 2 parts；
2) 800g, 5min are centrifuged, and abandon supernatant, and PBS is washed three times；
3) 500 μ L Hepes-Tyrodes buffer (0.8mmol/L calcium chloride) and Hepes-Tyrodes are used respectively
Again suspend buffer (high potassium) blood platelet, dilution blood platelet to about 5~10 × 109/ L, difference flag F and F0；
4) to F0The short bacterium bar peptide solution of final concentration of 5 μ g/L is added in streaming pipe, cell is depolarized completely；5) to F
And F0Streaming pipe adds final concentration of 10nmol/L DiBAC4 (3) solution (DMSO concentration is no more than 0.1%), gently shakes, mixes
It is even；Darkroom room temperature dyeing 60min；
6) machine testing in streaming.Voltage is set：FSC：E-1；SSC：313；FL1：555；FL2：150；FL3：150.
According to revised Nernst equation：V=Edye=-(RT/ZF) * ln (F/F0), F0Cell is represented to depolarize completely
When fluorescent value, F represents the fluorescent value that cell is under quiescent condition, calculates blood platelet membrane potential.
3 experimental results
To obtain 15 person-portion platelet membrane current potential average values be -55.9 ± 7mV for detection, with the patch-clamp method such as Lawrence T.
The result (- 60mV) that the result (- 52 ± 8mV) and Maclntyre done are done is similar, illustrates that the method measure blood of the present invention is small
The accuracy of plate film potential is very high.
The methodological study of the blood platelet membrane potential detection method of the present invention of embodiment 6
Examine the repeatability of the blood platelet membrane potential detection method of embodiment 1-5.Specific method is：According to embodiment 1-
5 either method detect the blood platelet membrane potential of 3 different peoples, each to detect 5 times, calculate the average value of blood platelet membrane potential
And variance.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.The result shows that this
Repeatability is good for the blood platelet membrane potential detection method of invention.
The Repeatability checking result of 1 embodiment 1 of table
| Different person-portions | 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 |
| Average value | -58.66 | -55.8 | -58.68 |
| Variance | 3.28 | 2.37 | 2.96 |
The Repeatability checking result of 2 embodiment 2-5 of table
Comparative example 1
1 experiment material and instrument
Fresh blood platelet, is put into blood platelet storage bag on the day of collection, and 22 DEG C of concussions preserve.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (being purchased from Sigma)
It is dissolved in the working solution that dimethyl sulfoxide (DMSO) is configured to 10 μm of ol/L, -20 DEG C of preservations.
Hepes-Tyrodes buffer (no calcium)：137mmol/L sodium chloride, 2.7mmol/L potassium chloride, 12mmol/L carbon
Sour hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/
L sodium hydroxides adjustment pH is 7.4.
Blood platelet fixer：4% paraformaldehyde and 2% glutaraldehyde are prepared using PBS.
Flow cytometer (BD, FACSCalibur)；Centrifuge (Eppendorf, 5154D).
2 experimental procedures
1) 500 μ L Single-donor platelets stostes, 800g, 5min centrifugation are taken；Supernatant is abandoned, PBS is washed three times.0.01% glutaraldehyde
+ 2% paraformaldehyde fixed under the conditions of 4 DEG C 2 it is small when, 800g, 5min centrifugation, Hepes-Tyrodes buffer (no calcium) rinse
Three times.Hepes-Tyrodes buffer (adding final concentration of 1.8mmol/L calcium chloride) dilute blood platelet to about 5~10 ×
109/ L, labeled as F0；
2) 500 μ L Single-donor platelets stostes, 800g, 5min centrifugation are taken；Supernatant is abandoned, PBS is washed three times.Hepes-
Tyrodes buffer (no calcium) are rinsed three times.Hepes-Tyrodes buffer (add final concentration of 1.8mmol/L chlorinations
Calcium) suspend blood platelet again；Blood platelet is diluted to about 5~10 × 109/ L, labeled as F；
3) to F and F0Streaming pipe adds final concentration of 100nmol/L DiBAC4 (3) solution, and (DMSO concentration is no more than
0.1%), gently shake, mix.Darkroom room temperature dyeing 20min；Machine testing in streaming, voltage are set：FSC：E-1；SSC：313；
FL1：555；FL2：150；FL3：150.
The blood platelet membrane potential of 3 different peoples is detected according to the method described above, it is each to detect 5 times, calculate blood platelet cross-film
The average value and variance of current potential, examine the repeatability of this method.
3 experimental results
Glutaraldehyde and the Repeatability checking of paraformaldehyde fixation the results are shown in Table 3.The result shows that the repeatability of this method is shown
Write the method for being worse than the present invention.
3 glutaraldehyde of table and the Repeatability checking result of paraformaldehyde fixation
| Different person-portions | 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 |
| Average value | -49.2 | -46.6 | -44.1 |
| Variance | 10.5 | 12.7 | 11.8 |
Comparative example 2
1 experiment material and instrument
Fresh blood platelet, is put into blood platelet storage bag on the day of collection, and 22 DEG C of concussions preserve.
Bis-oxonol (being purchased from Sigma) is dissolved in the working solution 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) is configured to 2mg/mL's using dimethyl sulfoxide (DMSO)
Working solution, -20 DEG C of preservations.
Hepes-Tyrodes buffer (no calcium)：137mmol/L sodium chloride, 2.7mmol/L potassium chloride, 12mmol/L carbon
Sour hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/
L sodium hydroxides adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium)：2.7mmol/L sodium chloride, 137mmol/L potassium chloride, 12mmol/L carbon
Sour hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/
L sodium hydroxides adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur)；Centrifuge (Eppendorf, 5154D).
2 experimental procedures
1) 1000 μ L Single-donor platelets stostes are taken, are divided into 2 parts；
2) 800g, 5min are centrifuged, and abandon supernatant, and PBS is washed three times；
3) 500 μ L Hepes-Tyrodes buffer (1.8mmol/L calcium chloride) and Hepes-Tyrodes are used respectively
Again suspend buffer (high potassium) blood platelet, dilution blood platelet to about 5~10 × 109/ L, difference flag F and F0；
4) to F0The short bacterium bar peptide solution of final concentration of 1 μ g/L is added in streaming pipe, cell is depolarized completely；
5) to F and F0Streaming pipe adds final concentration of 100nmol/L Bis-oxonol solution, and (DMSO concentration is no more than
0.1%), gently shake, mix；Darkroom room temperature dyeing 20min；
6) machine testing in streaming.Voltage is set：FSC：E-1；SSC：313；FL1：555；FL2：150；FL3：150.
According to revised Nernst equation：V=Edye=-(RT/ZF) * ln (F/F0), F0Cell is represented to depolarize completely
When fluorescent value, F represents the fluorescent value that cell is under quiescent condition, calculates blood platelet membrane potential.
The blood platelet membrane potential of 3 different peoples is detected according to the method described above, it is each to detect 5 times, calculate blood platelet cross-film
The average value and variance of current potential, examine the repeatability of this method.
3 experimental results
To obtain 15 person-portion platelet membrane current potential average values be -45.8 ± 10mV for detection, with the patch-clamp method such as Lawrence T.
Result (- 60mV) the difference that the result (- 52 ± 8mV) and Maclntyre done are done is larger, illustrates that this method measures blood platelet
The accuracy of film potential is general.
The Repeatability checking of this method the results are shown in Table 4.The result shows that the repeatability of this method is significantly worse than the side of the present invention
Method.
The Repeatability checking result of 4 the present embodiment method of table
| Different person-portions | 1(mV) | 2(mV) | 3(mV) |
| Average value | -43.8 | -41.7 | -49.2 |
| Variance | 9.8 | 10.7 | 10.4 |
Comparative example 3
1 experiment material and instrument
Fresh blood platelet, is put into blood platelet storage bag on the day of collection, and 22 DEG C of concussions preserve.
Bis (1,3-dibutylbarbituric acid) trimethineoxonol, DiBAC4 (3) (being purchased from Sigma)
It is dissolved in the working solution 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) is configured to 2mg/mL's using dimethyl sulfoxide (DMSO)
Working solution, -20 DEG C of preservations.
Hepes-Tyrodes buffer (no calcium)：133mmol/L sodium chloride, 6mmol/L potassium chloride, 12mmol/L carbonic acid
Hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L
Sodium hydroxide adjustment pH is 7.4.
Hepes-Tyrodes buffer (high potassium)：5mmol/L sodium chloride, 120mmol/L potassium chloride, 12mmol/L carbonic acid
Hydrogen sodium, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L glucose, 10mmol/L Hepes, 1mol/L
Sodium hydroxide adjustment pH is 7.4.
Flow cytometer (BD, FACSCalibur)；Centrifuge (Eppendorf, 5154D).
2 experimental procedures
7) 1000 μ L Single-donor platelets stostes are taken, are divided into 2 parts；
8) 800g, 5min are centrifuged, and abandon supernatant, and PBS is washed three times；
9) 500 μ L Hepes-Tyrodes buffer (5mmol/L calcium chloride) and Hepes-Tyrodes are used respectively
Again suspend buffer (high potassium) blood platelet, dilution blood platelet to about 5~10 × 109/ L, difference flag F and F0；
10) to F0The short bacterium bar peptide solution of final concentration of 0.1 μ g/L is added in streaming pipe, cell is depolarized completely；
11) to F and F0Streaming pipe adds final concentration of 220nmol/L DiBAC4 (3) solution, and (DMSO concentration is no more than
0.1%), gently shake, mix；Darkroom room temperature dyeing 15min；
12) machine testing in streaming.Voltage is set：FSC：E-1；SSC：313；FL1：555；FL2：150；FL3：150.
According to revised Nernst equation：V=Edye=-(RT/ZF) * ln (F/F0), F0Cell is represented to depolarize completely
When fluorescent value, F represents the fluorescent value that cell is under quiescent condition, calculates blood platelet membrane potential.
The blood platelet membrane potential of 3 different peoples is detected according to the method described above, it is each to detect 5 times, calculate blood platelet cross-film
The average value and variance of current potential, examine the repeatability of this method.
3 experimental results
To obtain 15 person-portion platelet membrane current potential average values be -53.6 ± 8mV for detection, with the patch-clamp method such as Lawrence T.
Result (- 60mV) obtained by the result (- 52 ± 8mV) and Maclntyre of gained is similar, illustrates this method measure platelet membrane electricity
The accuracy of position is higher.
The Repeatability checking of this method the results are shown in Table 5.The result shows that the repeatability of this method is significantly worse than the side of the present invention
Method.
The Repeatability checking result of 5 the present embodiment method of table
| Different person-portions | 1(mV) | 2(mV) | 3(mV) |
| Average 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 is noted that for the ordinary skill people of the art
Member, on the premise of the method for the present invention is not departed from, can also make some improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (10)
1. a kind of blood platelet membrane potential detection method, it is characterised in that comprise the following steps：
A) blood platelet bulk samples centrifuge, and abandon supernatant, wash；
B) platelet sample for taking part to be handled through step a), is 1- using calcium chloride containing 0.8-5mmol/L and KCE content
The Hepes-Tyrodes buffer of 6mmol/L suspend blood platelet again, dilution blood platelet to 5~10 × 109/ L, adds streaming
Pipe, is denoted as F；The platelet sample that part is handled through step a) separately is taken, the use of KCE content is 120-150mmol/L and chlorination
Sodium content is that the Hepes-Tyrodes buffer of 2-20mmol/L suspend blood platelet again, dilution blood platelet to 5~10 ×
109/ L, adds streaming pipe, is denoted as F0；
C) to F0The short bacterium bar peptide solution of final concentration of 0.1-5 μ g/L is added in streaming pipe, cell is depolarized completely；
D) to F and F0Streaming pipe adds final concentration of 10-200nmol/L DiBAC4 (3) solution, gently shakes, and mixes, darkroom room
Temperature dyeing 15-60min；
E) machine testing in streaming；
According to revised Nernst equation：V=Edye=-(RT/ZF) * ln (F/F0), blood platelet membrane potential is calculated, wherein,
F0Fluorescent value when cell depolarizes completely is represented, F represents the fluorescent value that cell is under quiescent condition.
2. blood platelet membrane potential detection method according to claim 1, it is characterised in that DiBAC4 (3) in step d)
The short final concentration of 0.5-2 μ g/L of bacterium bar peptide in final concentration of 80-100nmol/L, step c).
3. blood platelet membrane potential detection method according to claim 2, it is characterised in that DiBAC4 (3) in step d)
The short final concentration of 1 μ g/L of bacterium bar peptide in final concentration of 100nmol/L, step c).
4. blood platelet membrane potential detection method according to claim 1, it is characterised in that dyeing time is in step d)
15-22min。
5. blood platelet membrane potential detection method according to claim 4, it is characterised in that dyeing time is in step d)
20min。
6. blood platelet membrane potential detection method according to claim 1, it is characterised in that in step b), be denoted as the blood of F
The platelet sample KCE content in Hepes-Tyrodes buffer used that suspends again is 1-5mmol/L, is denoted as F0Blood
The platelet sample KCE content in Hepes-Tyrodes buffer used that suspends again is 120-140mmol/L.
7. blood platelet membrane potential detection method according to claim 6, it is characterised in that in step b), be denoted as the blood of F
The platelet sample KCE content in Hepes-Tyrodes buffer used that suspends again is 2.7mmol/L, is denoted as F0Blood
The platelet sample KCE content in Hepes-Tyrodes buffer used that suspends again is 137mmol/L.
8. blood platelet membrane potential detection method according to claim 1, it is characterised in that in step b), be denoted as the blood of F
The platelet sample calcium chloride content in Hepes-Tyrodes buffer used that suspends again is 0.8-3mmol/L.
9. blood platelet membrane potential detection method according to claim 8, it is characterised in that in step b), be denoted as the blood of F
The platelet sample calcium chloride content in Hepes-Tyrodes buffer used that suspends again is 1.8mmol/L.
10. blood platelet membrane potential detection method according to claim 1, it is characterised in that the KCE content
Specifically contain the reagent of following concentration for the Hepes-Tyrodes buffer of 1-6mmol/L：137mmol/L sodium chloride,
2.7mmol/L potassium chloride, 12mmol/L sodium acid carbonates, 1mmol/L magnesium chlorides, 0.4mmol/L sodium dihydrogen phosphates, 5.5mmol/L
Glucose, 10mmol/L Hepes, pH 7.4.
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