CN105572018A - Method for measuring concentration of free magnesium ions in red cells by fluorescent indicators through being combined with flow cytometry - Google Patents
Method for measuring concentration of free magnesium ions in red cells by fluorescent indicators through being combined with flow cytometry Download PDFInfo
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- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 229910001425 magnesium ion Inorganic materials 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000000684 flow cytometry Methods 0.000 title claims abstract description 17
- 239000003269 fluorescent indicator Substances 0.000 title abstract 2
- 241001465754 Metazoa Species 0.000 claims abstract description 18
- 239000003814 drug Substances 0.000 claims abstract description 14
- 230000015654 memory Effects 0.000 claims abstract description 14
- 210000003743 erythrocyte Anatomy 0.000 claims description 63
- 210000004369 blood Anatomy 0.000 claims description 38
- 239000008280 blood Substances 0.000 claims description 38
- 239000012530 fluid Substances 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 210000003462 vein Anatomy 0.000 claims description 9
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 8
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 229910001424 calcium ion Inorganic materials 0.000 claims description 8
- 239000012981 Hank's balanced salt solution Substances 0.000 claims description 6
- 239000003146 anticoagulant agent Substances 0.000 claims description 6
- 229940127219 anticoagulant drug Drugs 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 159000000003 magnesium salts Chemical class 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000011435 rock Substances 0.000 claims description 6
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 5
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- 229960002897 heparin Drugs 0.000 claims description 5
- 229920000669 heparin Polymers 0.000 claims description 5
- 210000000170 cell membrane Anatomy 0.000 claims description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 4
- NGCVJRFIBJVSFI-UHFFFAOYSA-I magnesium green Chemical compound [K+].[K+].[K+].[K+].[K+].C1=C(N(CC([O-])=O)CC([O-])=O)C(OCC(=O)[O-])=CC(NC(=O)C=2C=C3C(C4(C5=CC(Cl)=C([O-])C=C5OC5=CC([O-])=C(Cl)C=C54)OC3=O)=CC=2)=C1 NGCVJRFIBJVSFI-UHFFFAOYSA-I 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 4
- 238000005057 refrigeration Methods 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 230000012447 hatching Effects 0.000 claims description 3
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- 238000007865 diluting Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 239000011777 magnesium Substances 0.000 abstract description 85
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 abstract description 57
- 229910052749 magnesium Inorganic materials 0.000 abstract description 56
- 239000000523 sample Substances 0.000 abstract description 25
- 238000005259 measurement Methods 0.000 abstract description 16
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- 238000003745 diagnosis Methods 0.000 abstract 1
- 150000001455 metallic ions Chemical class 0.000 abstract 1
- 229940091250 magnesium supplement Drugs 0.000 description 55
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 241000700159 Rattus Species 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 9
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- 238000011084 recovery Methods 0.000 description 7
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 6
- 238000003860 storage Methods 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
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- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
- G01N2001/386—Other diluting or mixing processes
- G01N2001/388—Other diluting or mixing processes mixing the sample with a tracer
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- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Dispersion Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a method for measuring the concentration of free magnesium ions in red cells by fluorescent indicators through being combined with flow cytometry, and aims at precisely measuring the concentration level (RBC[Mg<2+>]i) of endogenous metallic ions of magnesium ions closely interrelated to great amount of life activities in the red cells in the free state. A magnesium ion fluorescent probe is used for combining the flow cytometry, and the method has the advantages that the measurement is fast and is in real time; the operation is simple and convenient; the sample collection flux is high; the measuring result is precise and stable, and the like. The RBC[Mg<2+>]i measured by the method provided by the invention can be used for effectively showing the magnesium loss of the animal body in the diet intake lack, diabetes and senility process, and can also be used for effectively showing the memory power level of old animals and predicting the medication effect of exogenous magnesium compensation treatment of old animal memory injury. The RBC[Mg<2+>]i measured by the method can be a potential clinic physical index, and the reference standard can be provided for the health diet structure conditioning, disease diagnosis and monitoring and individualized precise medical care medication.
Description
Technical field
The present invention relates to the method measuring free magnesium ion concentration in red blood cell with fluorescence indicator in conjunction with fluidic cell method.
Background technology
Life science fundamental research and clinical field have reported human body and animal endogenous content of magnesium and physiological and pathological process close ties.Measure biological endogenous property magnesium level to scientific research, clinical monitoring and medication with reference to there being important meaning.At present for the measurement of biological endogenous property magnesium level, varied in technological means, also multifarious on detection target spot, it is low all to there is degree of accuracy in the technology much reported, the problem that applicability is narrow.
Magnesium in biosome in each tissue, cell and body fluid can be divided into free magnesium and generally in conjunction with magnesium two class.Lot of documents report display: endocellular liberation magnesium directly combines the biomolecule such as such as ATP, enzyme, cell-membrane receptor, participates in regulating vital movement; The physiological and pathological development process that endocellular liberation magnesium is relevant to some magnesium metabolic disorders has more sign function.The such as free magnesium of brain, musculature has been used to study the adjoint magnesium of corresponding disease and has run off, but they exist application limitation.And blood magnesium component is exchanged by systemic blood circulation and magnesium ion, reconcile the magnesium level that body is respectively organized; The haemocyte magnesium that dissociates has and extensively characterizes body and respectively organize magnesium level, and indicates the potential ability of multiple physiological and pathological development process.
The technology of existing measurement endocellular liberation magnesium mainly contains nuclear magnetic resonance method and fluorescence indicator method.What nuclear magnetic resonance method essence was measured is endocellular phosphorus content, then converses magnesium ion content with the incorporating parametric of magnesium ion and phosphorous ATP, and be one very indirectly technology, disturbing factor is many; And nuclear magnetic resonance technique is mainly used in organizes aspect, lower to the Erythrocyte measure precision suspended; The blood sampling volume that its needs is also many, much larger than we technology (10mL contrasts 100 μ L) at present nuclear magnetic resonance method measure the erythrocyte magnesium that dissociates and be only applied to several routine diabetes, hypertensive magnesium Study on loss.Fluorescence indicator method adopts permeability cell probe directly in conjunction with endocellular liberation magnesium, and then produces fluorescence, and fluorescence intensity corresponding with free magnesium level height (see Fig. 2), precision is higher.In existing bibliographical information, fluorescence indicator fado for measure adhere-wall culture neural cellular in free magnesium ion concentration, have no for Erythrocyte measure.And neuron dissociate the fluoroscopic examination of magnesium and calibration process complicated, relate to professional image software and a large amount of manual handle, the large sample amount not being suitable for Red Blood Cells Suspension detects.In addition, the emission peak wavelength of the many fluorescence indicators in the past used moves along with the change of magnesium ion concentration, complex operation during measurement, also easily introduces error with during fluorescence intensity conversion magnesium ion actual concentration.Meanwhile, some technology adopt fluorescent microscope to detect fluorescence indicator, and this kind of fluorescent quantitation amount precision is low.
Summarize, the technology of existing measurement endocellular liberation magnesium exists that precision is low, sample requirements large, is not suitable for surveying red blood cell, the defects such as method of operating is loaded down with trivial details, disturbing factor is many.
Summary of the invention
In order to solve technical matters existing in background technology, the invention provides a kind of method measuring free magnesium ion concentration in red blood cell with fluorescence indicator in conjunction with Flow Cytometry.
Technical solution of the present invention:
Measure the method for free magnesium ion concentration in red blood cell in conjunction with Flow Cytometry with fluorescence indicator, its special character is: comprise the following steps:
1] reagent prepares:
Physiological buffer (not calcic, magnesium), aseptic, refrigeration;
Magnesium salts;
Blood anticoagulant, refrigeration;
Cell-membrane permeable magnesium ion fluorescence indicator, freezing;
Cosolvent (PluronicF-127), room temperature;
Calcium ions and magnesium ions carrier, freezing;
2] blood is got:
Blood anticoagulant is added in EP pipe, after get animals iv blood and add in EP pipe along tube wall, mix in time, obtain whole blood sample stand-by;
3] sample preparation and hatching:
3.1] dilute magnesium salts to physiology magnesium ion concentration with hanks equilibrium liquid, be adjusted to physiological PH value, obtained working fluid, is put in room temperature for subsequent use;
3.2] by step 2] the whole blood sample step 3.1 that obtains] working fluid prepared is diluted to red blood cell concentration 3 ~ 9 × 10
9individual/about L, is put in room temperature for subsequent use;
3.3] taken out by freezing magnesium ion fluorescence indicator, hold to thawing, 1:1.25 adds cosolvent PluronicF-127 and mixes by volume wherein; Then mix with working fluid under the condition of lucifuge, and mix by vortex oscillator;
3.4] in EP pipe by 3.2] whole blood sample and step 3.3 after dilution] the obtained working fluid containing magnesium ion fluorescence indicator mixes, after put into water-bath, doing first time hatches, and rocks EP at interval of 6-8 minute and manage once;
3.5] after having hatched, in EP pipe, add working fluid, rock resuspended, rear room temperature is centrifugal;
3.6] take out supernatant with pipettor to discard, add working fluid to the erythroprecipitin at the bottom of pipe, jog EP manages resuspended; Or it is resuspended with the liquid transfer gun head pressure-vaccum of clip;
3.7] step 3.6 is performed again];
3.8] red blood cell after resuspended is put into water-bath and is carried out second time and hatch, and endoerythrocytic magnesium ion fluorescence indicator is fully combined with endocellular liberation magnesium ion;
3.9] hatch rear taking-up pipe for the second time to dry, put into the fluorescence intensities to be measured such as magazine;
4] free magnesium ion concentration typical curve in red blood cell is set up:
4.1] prepare with hanks equilibrium liquid and magnesium sulfate or magnesium chloride the magnesium ion concentration gradient solution meeting physiological range;
4.2] by step 3.9] red blood cell many parts of parallel sampleses of obtaining washing with hanks equilibrium liquid, rear with step 4.1] the magnesium ion concentration gradient solution prepared mixes, add calcium ions and magnesium ions carrier, the magnesium ion concentration of intra-erythrocyte is balanced in each pipe;
4.3] 37 DEG C of water-baths 1 ~ 3 hour, dry pipe after taking-up, put into the fluorescence intensities to be measured such as magazine;
4.4] with the fluorescence intensity Criterion curve of magnesium ion concentration gradient solution and counter sample;
5] with the fluorescence intensity level of measured by flow cytometry sample:
By 3.9] and 4.3] sample that obtains of step injects flow cytometer, in forward angle/lateral angle image, choose red blood cell quadrant, in fluorescence intensity histogram figure, choose fluorescence peak further, read its average fluorescent strength value;
6] by 5] obtain average fluorescent strength value substitute into 4.4] typical curve that obtains, calculate sample endocellular liberation magnesium ion concentration.
Fluorescence indicator is the Magnesiumgreen that Invitrogen company produces, its working concentration 2 ~ 10 μm of ol/L.
Above-mentioned animals iv blood is rat tail vein blood, mouse ball rear vein beard blood, people's ulnar vein blood or finger tip blood.
Step 3.2] in by step 2] the whole blood sample step 3.1 that obtains] to be diluted to red blood cell concentration be 5 ~ 7 × 10 for the working fluid prepared
9individual/L.
Diluting erythrocytic physiological buffer is hanks equilibrium liquid (HanksBalancedSalts, HBSS); Contained physiological concentration magnesium salts is magnesium sulfate or magnesium chloride.
Blood anticoagulant is heparin.
First time hatches, and bath temperature is 37 DEG C, 40 ~ 80 minutes.
Second time is hatched, and bath temperature is 37 DEG C, 25 ~ 50 minutes.
The calcium ions and magnesium ions carrier used is A23187, its working concentration 20 ~ 75 μm of ol/L.
Free magnesium ion improves the application in memory medicine in preparation.
The advantage that the present invention has:
1, the present invention is specially for magnesium ion content in the born of the same parents of this specific cells of measurement erythrocyte group, has and samples rapid, real-time, Wicresoft, simple and convenient advantage.The application of drain cell technology more ensure that high flux and the specificity of sample detection.
2, measuring accuracy of the present invention is high.With the recovery of measurement standard magnesium ion concentration sample of the present invention between 98% ~ 102%, the dispersion of duplicate measurements sample is no more than 2%, specifically as shown in table 1.
3, wide application of the present invention, has sign effect to multiple physiological and pathological process.Free magnesium ion concentration (RBC [Mg in the red blood cell using the present invention to measure
2+]
i), for monitoring the change of large mouse diet magnesium nutrition intake; Study the health magnesium loss situation of diabetic mice and compensate treatment containing magnesium medicine; Study the adjoint health magnesium loss situation of large mouse aging course and contain magnesium medicine to the treatment of senile rat failure of memory; Study the Body Magnesium loss situation of Alzheimer's disease model mice and contain magnesium medicine to the treatment etc. of its failure of memory.Be worth ben, the animal RBC [Mg measured by the present invention
2+]
idirectly can characterize large mouse memory level, this is the original important discovery of neuroscience field one.More than find that there is the key player helping Study on Endogenous magnesium and play the part of in physiological activity and some relevant disease Development process, and contain the mechanism of action of magnesium drug therapy relevant disease.We estimate that the scientific discovery on these large mouse will be equally applicable to human research, now carry out clinical research.Future clinical uses human body content of magnesium level of the present invention, by the conditioning for health diet structure, the early diagnosis of magnesium disappearance relevant disease and prevention, and the many-side such as precisely to treat containing the judgement of magnesium drug medication dosage and individuation effective normative reference is provided, having may as the potential of clinical physiological index.
Accompanying drawing explanation
Fig. 1 is the graphical analysis of flow cytometer, chooses the schematic diagram of quadrant and door;
Fig. 2 is the schematic diagram that magnesium ion concentration typical curve is set up;
Fig. 3 is the RBC [Mg that the present invention surveys
2+]
ithe scientific research result of effective reflection animal diet followed magnesium intake change;
Fig. 4 is the RBC [Mg that the present invention surveys
2+]
ithe scientific research result of effective reflection diabetes (high lipid food) model mice health magnesium wastage;
Fig. 5 is the RBC [Mg that the present invention surveys
2+]
ithe scientific research result of effective reflection animal health magnesium wastage in aging course;
Fig. 6 is the RBC [Mg that the present invention surveys
2+]
ithe scientific research result of Efficient Characterization animal memory (Tmaze experiment).
Embodiment
Embodiment 1:
1, reagent material
Hanks equilibrium liquid HanksBalancedSalts (HBSS, Sigma-Aldrich), not calcium-magnesium-containing ion, PH=7.4, aseptic, 4 DEG C of storages;
80mmol/LMgSO
4(being dissolved in deionized water), 4 DEG C of storages;
Heparin Heparin, 4 DEG C of storages;
Cell-membrane permeable magnesium ion fluorescence indicator Magnesiumgreen (MaG, Invitrogen) 1g/L is dissolved in DMSO, is packed as 10ul/ pipe ,-20 DEG C of storages;
Cosolvent PluronicF-127 (Invitrogen), room temperature storage;
Calcium ions and magnesium ions carrier A 23187 (Sigma-Aldrich) ,-20 DEG C of storages;
2, blood is got
Heparin is added in advance in 200uLEP pipe (be 1:20 ~ 1:50 with target blood sampling volume volume ratio), get animals iv blood (rat tail vein blood, mouse ball rear vein beard blood, people's ulnar vein blood or finger tip blood) add in EP pipe along tube wall, timely and soft mixing.
3, sample preparation and hatching:
With HBSS storing solution dilution 80mmol/LMgSO
4to 0.5mmol/L, regulate pH value to 7.4, use after being placed into room temperature, hereinafter referred to as " working fluid ".
In 2mLEP pipe, with containing 0.5mmol/LMgSO
4hBSS (working fluid) whole blood is diluted 1200 times, soft repeatedly put upside down pipe mixing, for subsequent use.Now erythrocyte is about 3 × 10
9individual/L, available cells tally is measured to determine.
Rapidly by fluorescence indicator MaG from-20 DEG C of taking-ups, hold thawing.Add F-127 wherein, addition is the corresponding 1.25 times of volume F-127 of 1 volume MaG volume.Mix rapidly with pipettor.By the dilution proportion of the corresponding 50 times of volume working fluids of 1 volume MaG, mix with vortex.This process need is tried one's best lucifuge.
The Magnesiumgreen measuring the blood sample after 180uL dilution and 60uL dilution adds 2mLEP pipe, and soft rocks pipe mixing, and puts into 37 DEG C of water-baths 1 hour.Hatch and within process every 6 ~ 8 minutes, rock EP pipe once, in case hemostasis cell settlement.
After 1 hour, take out pipe and dry.1700uL working fluid is added, cleaning fluorescent dye to every pipe.Gentle inversion mixes, and in 1000rpm room temperature centrifugal 3 minutes.Centrifugal complete, the taking-up supernatant careful with pipettor discards, and adds 180uL working fluid to the red blood cell at the bottom of pipe, and jog pipe is resuspended; Also can mix with the liquid transfer gun head pressure-vaccum of clip.After re-suspended cell, again clean once by above process and use 180uL working fluid resuspended.
Washed red blood cell is put back to 37 DEG C of water-baths and place 30 minutes, intracellular fluorescence indicator is fully combined with magnesium ion.
Hatch rear taking-up pipe to dry, put into magazine etc. to be measured.
4, flow cytomery
Sample uses BDFACSCalibur model flow cytomery.
As shown in Figure 1, Fig. 1 a, chooses red blood cell quadrant, is designated as R1 sampling process in forward angle and lateral angle (FSC/SSC) image; Fig. 1 b, adopt FL1 channel measurement fluorescence intensity, in FL1 histogram, x-axis represents fluorescence intensity (FL1), and y-axis represents the red blood cell number (counts) that each fluorescence intensity is corresponding.The door M1 of fluorescence peak is chosen based on R1 quadrant further in Fig. 1 a.Get 10000 cells in M1 door during statistics, read its average fluorescent strength.
5, magnesium ion actual concentrations calibration (foundation of typical curve)
With HBSS and the MgSO of not calcium-magnesium-containing ion
4prepare physiological range magnesium ion concentration gradient.
Red blood cell step 3 obtained many parts of parallel samples HBSS (not calcic, magnesium) wash 2 times, it are mixed with magnesium ion concentration gradient solution.In each pipe, add calcium ions and magnesium ions carrier A 23187 to final concentration 25 μm of ol/L, the magnesium ion concentration of intra-erythrocyte is balanced.Pipe, after 2 hours, is dried after taking-up by 37 DEG C of water-baths, measures fluorescence intensity.With standard model magnesium ion concentration gradient and corresponding fluorescence intensity Criterion curve.Be illustrated in figure 2 magnesium ion concentration canonical plotting: measure magnesium ion concentration gradient standard sample, take fluorescence intensity level as transverse axis, magnesium ion concentration is the longitudinal axis, does index return curve, calculated curve equation.The fluorescence intensity level of testing sample is substituted into curvilinear equation, tries to achieve the actual magnesium ion concentration of sample.
Fig. 3 is one of application example: free magnesium ion concentration RBC [Mg in the red blood cell of the present invention's actual measurement
2+]
i) effectively can reflect that animal diet followed magnesium intake changes.Mouse fed common magnesium feed (magnesium content 0.15%, mass percent) or magnesium-deficient diet (magnesium content 0.003%) after 4 weeks, low magnesium group RBC [Mg
2+]
idecline (t check, p<0.001, error line represent standard deviation (SD)) more remarkable in normal group.
Fig. 4 is application example two: the RBC [Mg of the present invention's actual measurement
2+]
ieffectively reflect the health magnesium wastage of diabetic mice, and the recovery effects that exogenous magnesium compensates.ICR mouse is respectively with normal diet (NC), high heat food (HC), after high heat food adds and feeds 12 months containing magnesium medicine (HC+Mg), high heat group than normal group fasting blood-glucose (as Fig. 4 a) and Diagnostic Value of Fasting Serum insulin (as Fig. 4 b) concentration significantly rise (p<0.001), demonstrate high heat group and create insulin resistant and diabetes complications shape, model construction is successful; Now high heat group is than the RBC [Mg of normal group
2+]
i(as Fig. 4 c) significantly declines (p<0.01), demonstrates the magnesium loss that diabetic symptom causes mouse body.After high heat group mouse Mg supplementation is taken in, RBC [Mg
2+]
ithe decline of (as Fig. 4 c) obtains recovery, and now fasting blood-glucose (as Fig. 4 a) and Diagnostic Value of Fasting Serum insulin (as Fig. 4 b) all got back to normal level, demonstrate magnesium and supplement result for the treatment of to diabetic mice.(each figure medial error line represents standard deviation (SD))
Fig. 5 is application example three: the RBC [Mg of the present invention's actual measurement
2+]
ieffectively reflect the health magnesium wastage of animal in aging course.RBC [the Mg of statistics Different Month ICR female mice
2+]
i.Take monthly age as transverse axis, RBC [Mg
2+]
ifor longitudinal axis mapping, can find in aging course, there is RBC [Mg in Female ICR mice
2+]
iwith the monthly age decline situation, wherein 2.5 monthly ages, 3 the monthly age mouse respectively with 18 the monthly age mouse there were significant differences; Prompting RBC [Mg
2+]
ican run off by the old and feeble health magnesium caused of reflection.
Fig. 6 is application example four: the RBC [Mg of the present invention's actual measurement
2+]
ienergy Efficient Characterization animal memory level, and can predict containing magnesium medicine the recovery effects of senile rat memory injury.Detect the RBC [Mg of Old Male SD rat
2+]
iwith spatial memory (T maze experiment).Fig. 6 a, with RBC [Mg
2+]
ifor transverse axis, spatial memory must be divided into the longitudinal axis to map.Visible magnesium ion concentration and the linear positive correlation (R of spatial memory
2=0.64, p=0.0006); Prompting RBC [Mg
2+]
ithe memory level of geriatric animals individuality can be characterized.Fig. 6 b, Fig. 6 c, give after medicine magnesium supplements to senile rat, the amplitude that animal individual memory promotes is different, basic RBC [Mg
2+]
ilower individuality, the difference (after administration, behavioral experiment score deducts before administration) that before and after administration, memory promotes is larger; Display RBC [Mg
2+]
ithe effect recovering senile rat memory injury containing magnesium medicine can be predicted.
Embodiment 2:
Table 1: the present invention measures RBC [Mg
2+]
iquality inspection (recovery and dispersion).The red blood cell sample (sequence number 1 ~ 3) of preparation standard physiology magnesium ion concentration, detects its actual concentrations by this method, calculates the recovery; The average recovery rate of visual standard sample is between 98% ~ 102%, and measuring technique has well reappeared the actual value of standard model, the accuracy of display this method.Sequence number 4 and 5 is the measurements to actual animal blood sample, and wherein No. 4 is a young rats, and No. 5 is an aged rat; The dispersion of visible every animal duplicate measurements three times (A, B, C), all below 2%, shows the stability of this method; Measurement result young rats average is 0.27mmol/L simultaneously, and aged rat average is 0.20mmol/L, and demonstrate old and feeble adjoint magnesium and run off, this finds it is consistent with previous literature and our research.
Table 1RBC [Mg
2+]
ithe quality control of measuring technique
Claims (10)
1. measure the method for free magnesium ion concentration in red blood cell with fluorescence indicator in conjunction with Flow Cytometry, it is characterized in that: comprise the following steps:
1] reagent prepares:
Physiological buffer, aseptic, refrigeration;
Magnesium salts;
Blood anticoagulant, refrigeration;
Cell-membrane permeable magnesium ion fluorescence indicator, freezing;
Cosolvent (PluronicF-127), room temperature;
Calcium ions and magnesium ions carrier, freezing;
2] blood is got:
Blood anticoagulant is added in EP pipe, after get animals iv blood and add in EP pipe along tube wall, mix in time, obtain whole blood sample stand-by;
3] sample preparation and hatching:
3.1] dilute magnesium salts to physiology magnesium ion concentration with hanks equilibrium liquid, be adjusted to physiological PH value, obtained working fluid, is put in room temperature for subsequent use;
3.2] by step 2] the whole blood sample step 3.1 that obtains] working fluid prepared is diluted to red blood cell concentration 3 ~ 9 × 10
9individual/about L, is put in room temperature for subsequent use;
3.3] taken out by freezing magnesium ion fluorescence indicator, hold to thawing, 1:1.25 adds cosolvent PluronicF-127 and mixes by volume wherein; Then mix with working fluid under the condition of lucifuge, and mix by vortex oscillator;
3.4] in EP pipe by 3.2] whole blood sample and step 3.3 after dilution] the obtained working fluid containing magnesium ion fluorescence indicator mixes, after put into water-bath, doing first time hatches, and rocks EP at interval of 6-8 minute and manage once;
3.5] after having hatched, in EP pipe, add working fluid, rock resuspended, rear room temperature is centrifugal;
3.6] take out supernatant with pipettor to discard, add working fluid to the erythroprecipitin at the bottom of pipe, jog EP manages resuspended; Or it is resuspended with the liquid transfer gun head pressure-vaccum of clip;
3.7] step 3.6 is performed again];
3.8] red blood cell after resuspended is put into water-bath and is carried out second time and hatch, and endoerythrocytic magnesium ion fluorescence indicator is fully combined with endocellular liberation magnesium ion;
3.9] hatch rear taking-up pipe for the second time to dry, put into the fluorescence intensities to be measured such as magazine;
4] free magnesium ion concentration typical curve in red blood cell is set up:
4.1] prepare with hanks equilibrium liquid and magnesium sulfate or magnesium chloride the magnesium ion concentration gradient solution meeting physiological range;
4.2] by step 3.9] red blood cell many parts of parallel sampleses of obtaining washing with hanks equilibrium liquid, rear with step 4.1] the magnesium ion concentration gradient solution prepared mixes, add calcium ions and magnesium ions carrier, the magnesium ion concentration of intra-erythrocyte is balanced in each pipe;
4.3] 37 DEG C of water-baths 1 ~ 3 hour, dry pipe after taking-up, put into the fluorescence intensities to be measured such as magazine;
4.4] with the fluorescence intensity Criterion curve of magnesium ion concentration gradient solution and counter sample;
5] with the fluorescence intensity level of measured by flow cytometry sample:
By 3.9] and 4.3] sample that obtains of step injects flow cytometer, in forward angle/lateral angle image, choose red blood cell quadrant, in fluorescence intensity histogram figure, choose fluorescence peak further, read its average fluorescent strength value;
6] by 5] obtain average fluorescent strength value substitute into 4.4] typical curve that obtains, calculate sample endocellular liberation magnesium ion concentration.
2. the method measuring free magnesium ion concentration in red blood cell with fluorescence indicator in conjunction with Flow Cytometry according to claim 1, it is characterized in that: fluorescence indicator is the Magnesiumgreen that Invitrogen company produces, its working concentration 2 ~ 10 μm of ol/L.
3. the method measuring free magnesium ion concentration in red blood cell with fluorescence indicator in conjunction with Flow Cytometry according to claim 1, is characterized in that: described animals iv blood is rat tail vein blood, mouse ball rear vein beard blood, people's ulnar vein blood or finger tip blood.
4., according to the arbitrary described method measuring free magnesium ion concentration in red blood cell with fluorescence indicator in conjunction with Flow Cytometry of claim 1-3, it is characterized in that: step 3.2] in by step 2] the whole blood sample step 3.1 that obtains] to be diluted to red blood cell concentration be 5 ~ 7 × 10 for the working fluid prepared
9individual/L.
5. according to the arbitrary described method measuring free magnesium ion concentration in red blood cell with fluorescence indicator in conjunction with Flow Cytometry of claim 1-3, it is characterized in that: diluting erythrocytic physiological buffer is hanks equilibrium liquid (HanksBalancedSalts, HBSS); Contained physiological concentration magnesium salts is magnesium sulfate or magnesium chloride.
6., according to the arbitrary described method measuring free magnesium ion concentration in red blood cell with fluorescence indicator in conjunction with Flow Cytometry of claim 1 and 3, it is characterized in that: blood anticoagulant is heparin.
7. the method measuring free magnesium ion concentration in red blood cell with fluorescence indicator in conjunction with Flow Cytometry according to claim 1, is characterized in that: first time hatches, and bath temperature is 37 DEG C, 40 ~ 80 minutes.
8. the method measuring free magnesium ion concentration in red blood cell with fluorescence indicator in conjunction with Flow Cytometry according to claim 1, is characterized in that: second time is hatched, and bath temperature is 37 DEG C, 25 ~ 50 minutes.
9. the method measuring free magnesium ion concentration in red blood cell with fluorescence indicator in conjunction with Flow Cytometry according to claim 1, is characterized in that: the calcium ions and magnesium ions carrier of use is A23187, its working concentration 20 ~ 75 μm of ol/L.
10. free magnesium ion improves the application in memory medicine in preparation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111103272A (en) * | 2018-10-26 | 2020-05-05 | 山东大学 | Real-time screening and measuring system and method for cell specific photosensitive effect |
| CN111157501A (en) * | 2020-01-07 | 2020-05-15 | 香港科技大学深圳研究院 | Method for quantitatively measuring intracellular nano silver and silver ions |
| CN112285079A (en) * | 2020-10-19 | 2021-01-29 | 厦门大学 | Food nutrition absorption utilization evaluation method using sialic acid as mark and application |
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| US4336029A (en) * | 1980-08-15 | 1982-06-22 | Ortho Diagnostic Systems Inc. | Method and reagents for quantitative determination of reticulocytes and platelets in whole blood |
| CN1309968A (en) * | 2001-02-13 | 2001-08-29 | 暨南大学 | Pharmaceutical application of froctose biphosphate magnesium |
| US20040044228A1 (en) * | 2000-07-28 | 2004-03-04 | Koji Suzuki | Fluorescent probe for magnesium ion determination |
| CN101210930A (en) * | 2006-12-27 | 2008-07-02 | 中国科学院沈阳应用生态研究所 | Method for determining magnesium content in blood serum |
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| US4336029A (en) * | 1980-08-15 | 1982-06-22 | Ortho Diagnostic Systems Inc. | Method and reagents for quantitative determination of reticulocytes and platelets in whole blood |
| US20040044228A1 (en) * | 2000-07-28 | 2004-03-04 | Koji Suzuki | Fluorescent probe for magnesium ion determination |
| CN1309968A (en) * | 2001-02-13 | 2001-08-29 | 暨南大学 | Pharmaceutical application of froctose biphosphate magnesium |
| CN101210930A (en) * | 2006-12-27 | 2008-07-02 | 中国科学院沈阳应用生态研究所 | Method for determining magnesium content in blood serum |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111103272A (en) * | 2018-10-26 | 2020-05-05 | 山东大学 | Real-time screening and measuring system and method for cell specific photosensitive effect |
| CN111157501A (en) * | 2020-01-07 | 2020-05-15 | 香港科技大学深圳研究院 | Method for quantitatively measuring intracellular nano silver and silver ions |
| CN112285079A (en) * | 2020-10-19 | 2021-01-29 | 厦门大学 | Food nutrition absorption utilization evaluation method using sialic acid as mark and application |
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