CN115219311A - Detection test method for glucose uptake rate - Google Patents
Detection test method for glucose uptake rate Download PDFInfo
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- CN115219311A CN115219311A CN202210793774.9A CN202210793774A CN115219311A CN 115219311 A CN115219311 A CN 115219311A CN 202210793774 A CN202210793774 A CN 202210793774A CN 115219311 A CN115219311 A CN 115219311A
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- 230000004190 glucose uptake Effects 0.000 title claims abstract description 19
- 238000001514 detection method Methods 0.000 title claims abstract description 16
- 238000010998 test method Methods 0.000 title abstract description 6
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 47
- 210000004369 blood Anatomy 0.000 claims abstract description 25
- 239000008280 blood Substances 0.000 claims abstract description 25
- 101100393884 Drosophila melanogaster Glut1 gene Proteins 0.000 claims abstract description 21
- 101150058068 SLC2A1 gene Proteins 0.000 claims abstract description 21
- 238000000926 separation method Methods 0.000 claims abstract description 17
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 14
- 239000008103 glucose Substances 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 11
- 230000008727 cellular glucose uptake Effects 0.000 claims abstract description 6
- 238000007405 data analysis Methods 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims description 21
- 210000004027 cell Anatomy 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 13
- 238000005119 centrifugation Methods 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 10
- 238000012360 testing method Methods 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims 2
- 238000007865 diluting Methods 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 abstract description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 239000002609 medium Substances 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
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- 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
- G01N1/34—Purifying; Cleaning
-
- 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
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
-
- 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
-
- 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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Dispersion Chemistry (AREA)
- Urology & Nephrology (AREA)
- Diabetes (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a test method for detecting glucose uptake rate, which comprises the following four steps of red blood cell separation, red blood cell glucose uptake detection, flow detection Glut1 and data analysis, detects the glucose content in a whole blood sample, judges whether the glucose content is positive or not, has clear steps in the whole operation process, is mainly operated by centrifugal separation and the addition of various reagents in proportion, is convenient to operate and requires less time; the glucose uptake rate in blood can be better detected, and whether the patient suffers from diseases or not can be conveniently judged.
Description
Technical Field
The invention relates to the technical field of glucose uptake rate detection tests, in particular to a glucose uptake rate detection test method.
Background
The sugar in blood is called blood sugar, and is glucose in most cases, most of the energy required by the activity of each tissue cell in the body comes from glucose, so the blood sugar must be kept at a certain level to meet the requirement of each organ and tissue in the body, and the detection of the glucose uptake rate can detect various diseases, thereby having important clinical significance.
Disclosure of Invention
The present invention is directed to a method for testing glucose uptake, which solves the above problems of the prior art.
In order to achieve the purpose, the invention provides the following technical scheme: a test method for detecting glucose uptake rate comprises the following steps:
s1, separating red blood cells;
s2 glucose uptake detection by erythrocytes;
s3, detecting Glut1 in a flow mode;
and (s 4) analyzing data.
Preferably, the first step of red blood cell separation: mixing the anticoagulated whole blood sample with a diluent according to the volume ratio of 1:1 to obtain a diluted blood sample, adding a separation liquid which is equal to the diluted blood sample into the sterile silicified centrifuge tube, sucking the diluted blood sample by a suction tube, adding the diluted blood sample onto the liquid surface of the separation liquid of the sterile silicified centrifuge tube, and centrifuging for 15min at 900 g; centrifuging, dividing the centrifuge tube into four layers from top to bottom, discarding the first three layers, adding 10ml of cleaning solution into the remaining fourth layer of red blood cell layer, and centrifuging for 10min at 250 g; centrifuging, removing supernatant, resuspending the obtained cells with 5ml of washing solution, and centrifuging for 10min at 250 g; and repeating the centrifugation and discarding the supernatant, carrying out the centrifugation after the resuspension of the cleaning solution, discarding the supernatant after the centrifugation, discarding the supernatant, then carrying out the resuspension of the cells by using the PBS buffer solution with the volume of the original whole blood, and separating to obtain the red blood cells.
Preferably, the amount of the separation liquid should not be less than 3ml at minimum.
Preferably, the second step of the glucose uptake detection of erythrocytes: adding 1000 μ l of separated red blood cells into 1% concentrated serum, culturing overnight, and centrifuging at 250g for 5min; centrifuging, removing supernatant, adding 3000 μ L culture medium containing 5mmol/L glucose and 10% serum, and culturing at 37 deg.C and 5% CO2 for 1 hr to obtain test group and control group without erythrocyte; and centrifuging the test group and the control group at 250g for 10min, collecting supernatant, and detecting the content of glucose in the collected supernatant.
Preferably, the red blood cell glucose uptake rate is obtained by the following formula:
preferably, the third step is to flow-detect Glut1: taking 100 μ l of separated red blood cells, adding 10 μ l of Human Glut1 fluoroescein-conjugated Antibody, and reacting for 30min at room temperature in a dark place; after reaction, 100 μ l PBS buffer solution is added and mixed evenly, cells are collected by a flow cytometer, the fluorescence channel is FL 1-H, and the cell collection amount is 150000/case; the mean fluorescence intensity of Glut1 in total cells and the percentage gated on isotype control antibody were analyzed by CellQuest software.
Preferably, the average fluorescence intensity and the red blood cell uptake rate of Glut1 in red blood cells of 44 healthy control samples are calculated, and when the average fluorescence intensity and the red blood cell uptake rate of Glut1 are less than 75% of the average value, the sample is determined to be a positive sample.
Compared with the prior art, the invention has the beneficial effects that:
through four steps of red blood cell separation, red blood cell glucose uptake detection, flow detection Glut1 and data analysis, the glucose content in the whole blood sample is detected, whether the glucose content is positive or not is judged, the steps of the whole operation process are clear, the operation mainly comprises centrifugal separation and the proportional addition of various reagents, the operation is convenient, and the required time is short.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a test method for detecting glucose uptake rate comprises the following steps:
s1, separating red blood cells;
s2 glucose uptake detection by erythrocytes;
s3, detecting Glut1 in a flow mode;
and (s 4) analyzing data.
Specifically, the first step of red blood cell isolation: mixing the anticoagulated whole blood sample with a diluent according to the volume ratio of 1:1 to obtain a diluted blood sample, adding a separation liquid which is equal to the diluted blood sample into the sterile silicified centrifuge tube, sucking the diluted blood sample by a suction tube, adding the diluted blood sample onto the liquid surface of the separation liquid of the sterile silicified centrifuge tube, and centrifuging for 15min at 900 g; the centrifugal tube is divided into four layers from top to bottom after centrifugation, wherein the first layer is a plasma layer, the second layer is an annular milky white lymphocyte layer, the third layer is a transparent separation liquid layer, and the fourth layer is a red blood cell layer; carefully sucking a first layer of plasma layer, a second layer of annular milky white lymphocyte layer and a third layer of transparent separation liquid layer by using a suction pipe, discarding the first three layers, adding 10ml of cleaning solution into the remaining fourth layer of erythrocyte layer, and centrifuging for 10min at 250 g; centrifuging, removing supernatant, resuspending the obtained cells with 5ml of washing solution, and centrifuging for 10min at 250 g; and repeating the centrifugation and discarding the supernatant, carrying out the centrifugation after the resuspension of the cleaning solution, discarding the supernatant after the centrifugation, discarding the supernatant, then carrying out the resuspension of the cells by using the PBS buffer solution with the volume of the original whole blood, and separating to obtain the red blood cells.
Specifically, the amount of the separating medium to be used is controlled so that the amount of the separating medium to be used is not less than 3ml at the minimum.
Specifically, the second step of glucose uptake detection by erythrocytes: adding 1000 μ l of separated red blood cells into 1% concentrated serum, culturing overnight, and centrifuging at 250g for 5min; centrifuging, removing supernatant, adding 3000 μ L culture medium containing 5mmol/L glucose and 10% serum, and culturing at 37 deg.C and 5% CO2 for 1 hr to obtain test group and control group without erythrocyte; and centrifuging the test group and the control group at 250g for 10min, collecting supernatant, and detecting the content of glucose in the collected supernatant.
Specifically, the red blood cell glucose uptake rate is calculated by the following formula:
specifically, the third step is to flow-detect Glut1: taking 100 mul of separated red blood cells, adding 10 mul of Human Glut1 fluoroescein-conjugated Antibody, specifically a Human red blood cell separating medium kit, and reacting for 30min at room temperature in a dark place; adding 100 mu l of PBS buffer solution after reaction, uniformly mixing, collecting cells by using a flow cytometer, wherein the fluorescence channel is FL 1-H, and the quantity of the collected cells is 150000 per case; the mean fluorescence intensity of Glut1 in total cells and the percentage gated on isotype control antibody were analyzed by CellQuest software.
Specifically, the mean fluorescence intensity and the red blood cell uptake rate of Glut1 in red blood cells of 44 healthy control samples were calculated, and when the mean fluorescence intensity and the red blood cell uptake rate of Glut1 were less than 75% of the mean value, it was determined to be a positive sample.
The test instrument:
the adopted centrifugal machine is specifically a table type low-speed centrifugal machine, and the model is TD4A;
the model of the flow cytometer used was Calibur type II.
The working principle is as follows: the method comprises four steps of red blood cell separation, red blood cell glucose uptake detection, flow detection Glut1 and data analysis, detects the glucose content in the whole blood sample, and judges whether the glucose content is positive.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A method for detecting and testing glucose uptake rate, comprising the steps of:
s1, separating red blood cells;
s2 glucose uptake detection by erythrocytes;
s3, detecting Glut1 in a flow mode;
and (s 4) analyzing data.
2. The method according to claim 1, wherein the first step of red blood cell separation comprises: diluting an anticoagulated whole blood sample to obtain a diluted blood sample, adding a separation liquid which is equal to the diluted blood sample into a sterile silicification centrifuge tube, sucking the diluted blood sample by a suction tube, adding the blood sample onto the liquid surface of the separation liquid of the sterile silicification centrifuge tube, and centrifuging for 15min at 900 g; dividing the centrifugal tube into four layers from top to bottom after centrifugation, discarding the first three layers, adding 10ml of cleaning solution into the remaining fourth layer of erythrocyte layer, and centrifuging for 10min at 250 g; centrifuging, removing supernatant, resuspending the obtained cells with 5ml of washing solution, and centrifuging for 10min at 250 g; and repeating the centrifugation and discarding the supernatant, carrying out the centrifugation after the resuspension of the cleaning solution, discarding the supernatant after the centrifugation, discarding the supernatant, then carrying out the resuspension of the cells by using the PBS buffer solution with the volume of the original whole blood, and separating to obtain the red blood cells.
3. The method according to claim 2, wherein the assay comprises: the amount of the separation liquid should not be less than 3ml at least.
4. The method according to claim 1, wherein the second step of detecting glucose uptake by red blood cells comprises: adding 1000 μ l of separated red blood cells into 1% concentrated serum, culturing overnight, and centrifuging at 250g for 5min; centrifuging, removing supernatant, adding 3000 μ L culture medium containing 5mmol/L glucose and 10% serum, and culturing at 37 deg.C and 5% CO2 for 1 hr to obtain test group and control group without erythrocyte; and centrifuging the test group and the control group at 250g for 10min, collecting supernatant, and detecting the content of glucose in the collected supernatant.
5. The method according to claim 4, wherein the assay comprises: the red blood cell glucose uptake rate was calculated by the following formula:
6. the method for detecting glucose uptake rate according to claim 1, wherein the third step of detecting Glut1: adding 10 μ l Human Glut1 fluoroescein-conjugated Antibody into 100 μ l separated red blood cells, and reacting at room temperature in dark for 30min; after reaction, 100 μ l PBS buffer solution is added and mixed evenly, cells are collected by a flow cytometer, the fluorescence channel is FL 1-H, and the cell collection amount is 150000/case; the mean fluorescence intensity of Glut1 in total cells and the percentage gated on isotype control antibody were analyzed by CellQuest software.
7. The method for detecting glucose uptake rate according to claim 1, wherein the fourth step comprises data analysis: the mean fluorescence intensity and the red blood cell uptake rate of Glut1 in red blood cells of 44 healthy control samples were calculated, and when the mean fluorescence intensity and the red blood cell uptake rate of Glut1 were judged to be less than 75% of the mean value, the sample was determined to be positive.
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| CN202210793774.9A CN115219311A (en) | 2022-07-05 | 2022-07-05 | Detection test method for glucose uptake rate |
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| CN202210793774.9A CN115219311A (en) | 2022-07-05 | 2022-07-05 | Detection test method for glucose uptake rate |
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| CN202210793774.9A Pending CN115219311A (en) | 2022-07-05 | 2022-07-05 | Detection test method for glucose uptake rate |
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- 2022-07-05 CN CN202210793774.9A patent/CN115219311A/en active Pending
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