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Method for measuring concentration of free magnesium ions in red cells by fluorescent indicators through being combined with flow cytometry

A flow cytometry technology and a fluorescent indicator technology are applied in the field of measuring the concentration of free magnesium ions in red blood cells by combining the fluorescent indicator and the flow cytometry technology, which can solve the problems of cumbersome operation, low precision and cumbersome operation method, and achieve simple operation. Convenience, high measurement accuracy and wide application

Inactive Publication Date: 2016-05-11
康敏
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Problems solved by technology

In the existing literature reports, the fluorescent indicator method is mostly used to measure the intracellular free magnesium ion concentration of adherent cultured neurons, but it has not been used to measure red blood cells.
However, the fluorescence detection and calibration process of free magnesium in neurons is complicated, involving professional image processing software and a lot of manual processing, and is not suitable for the detection of large samples of suspended red blood cells.
In addition, the emission peak wavelength of many fluorescent indicators used in the past shifts with the change of magnesium ion concentration, the operation is cumbersome during measurement, and it is easy to introduce errors when converting the fluorescence intensity to the real concentration of magnesium ions
At the same time, some technologies use fluorescence microscopes to detect fluorescent indicators, which have low accuracy in quantitative quantification.
[0005] In summary, the existing techniques for measuring intracellular free magnesium have defects such as low precision, large sample demand, unsuitability for measuring red blood cells, cumbersome operation methods, and many interference factors.

Method used

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  • Method for measuring concentration of free magnesium ions in red cells by fluorescent indicators through being combined with flow cytometry
  • Method for measuring concentration of free magnesium ions in red cells by fluorescent indicators through being combined with flow cytometry
  • Method for measuring concentration of free magnesium ions in red cells by fluorescent indicators through being combined with flow cytometry

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Experimental program
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Effect test

Embodiment 1

[0056] 1. Reagent materials

[0057] Hanks Balanced Salts (HBSS, Sigma-Aldrich), without calcium and magnesium ions, pH=7.4, sterile, stored at 4°C;

[0058] 80mmol / LMgSO 4 (dissolved in deionized water), stored at 4°C;

[0059] Heparin, stored at 4°C;

[0060] Cell membrane permeable magnesium ion fluorescent indicator Magnesiumgreen (MaG, Invitrogen) 1g / L dissolved in DMSO, aliquoted into 10ul / tube, stored at -20°C;

[0061] Cosolvent PluronicF-127 (Invitrogen), stored at room temperature;

[0062] Calcium and magnesium ionophore A23187 (Sigma-Aldrich), stored at -20°C;

[0063] 2. Take blood

[0064] Add heparin into the 200uLEP tube in advance (the volume ratio of the target blood collection volume is 1:20~1:50), and take animal venous blood (rat tail venous blood, mouse retrobulbar venous plexus blood, human cubital venous blood or fingertip blood) ) into the EP tube along the tube wall, mix in time and gently.

[0065] 3. Sample processing and incubation:

[0066...

Embodiment 2

[0084] Table 1: The present invention measures RBC [Mg 2+ ] iQuality checks (recovery and dispersion). Prepare the erythrocyte sample (serial number 1~3) of standard physiological magnesium ion concentration, detect its actual concentration with this method, calculate recovery rate; The average recovery rate of visible standard sample is between 98%~102%, and measurement technique is very good weight The true value of the standard sample was found, showing the accuracy of the method. Nos. 4 and 5 are measurements of actual animal blood samples, wherein No. 4 is a young rat, and No. 5 is an old rat; it can be seen that the dispersion of three repeated measurements (A, B, C) of each animal is average Below 2%, the stability of this method is shown; the average value of the young rats measured at the same time is 0.27mmol / L, and the average value of the old rats is 0.20mmol / L, showing the loss of magnesium associated with aging, which is consistent with previous literature and ...

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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 [0001] The present invention relates to a method for measuring the concentration of free magnesium ions in red blood cells by combining a fluorescent indicator with a flow cytometry method. Background technique [0002] The basic research and clinical fields of life sciences have reported that the endogenous magnesium content of humans and animals is closely related to the physiological and pathological processes. Measurement of biological endogenous magnesium levels is of great significance for scientific research, clinical monitoring and drug reference. At present, the measurement of biological endogenous magnesium levels has various technical means and various detection targets. Many reported techniques have problems of low accuracy and narrow applicability. [0003] Magnesium in various tissues, cells and body fluids in organisms can be generally divided into two categories: free magnesium and bound magnesium. A large number of literature reports show t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14G01N1/28G01N1/38
CPCG01N15/1434G01N1/28G01N1/38G01N2001/388
Inventor 康敏
Owner 康敏
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