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Method for analyzing and evaluating erythropoiesis function of mouse marrow through flow cytometry

An analysis and evaluation, cytometer technology, applied in the field of flow cytometry, can solve the problems of inability to distinguish reticulocytes and mature red blood cells, unfavorable promotion and application, high price of antibodies, etc. The effect of detection time

Active Publication Date: 2016-07-06
江苏健易达医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It has been reported to use the mouse cell surface markers CD71 and Ter119 for dual-parameter labeling to study mouse erythroid cells, but this method cannot distinguish reticulocytes from mature erythrocytes, and the antibody price is generally high, so using both CD71 and Ter119 Antibodies make the detection cost high, which is not conducive to popularization and application

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  • Method for analyzing and evaluating erythropoiesis function of mouse marrow through flow cytometry

Examples

Experimental program
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Effect test

Embodiment 1

[0031] 1. Take BALB / c mouse bone marrow cells, count, and take 1 million cells;

[0032] 2. Add 1ml of thiazole orange staining solution to the mouse bone marrow cells, the concentration of the thiazole orange staining solution is 0.2 μg / ml, incubate at room temperature for 30 minutes, wash, centrifuge, and discard the supernatant;

[0033] 3. Add 0.1ml rat IgG for blocking, the concentration of the rat IgG is 0.1mg / ml, incubate at 4°C for 10min;

[0034] 4. Add 0.1ml of APCRatAnti-MouseTer119, the concentration of APCRatAnti-MouseTer119 is 4μg / ml, and incubate at 4°C for 30min;

[0035] 5. Add 0.1ml PBS, flow cytometry analysis, thiazole orange excitation light 488nm, emission light 530nm, APC excitation light 638nm, emission light 660nm;

[0036] 6. Collect 20,000 cells, and use flow cytometry software to analyze the cell distribution of fluorescent populations.

Embodiment 2

[0038] 1. Take BALB / c mouse bone marrow cells, count, and take 1 million cells;

[0039] 2. Add 0.1 ml of thiazole orange staining solution to the mouse bone marrow cells, the concentration of the thiazole orange staining solution is 1 μg / ml, incubate at room temperature for 5 minutes, wash, centrifuge, and discard the supernatant;

[0040] 3. Add 0.1ml rat IgG for blocking, the concentration of the rat IgG is 0.1mg / ml, incubate at 4°C for 10min;

[0041] 4. Add 0.1ml of APCRatAnti-MouseTer119, the concentration of APCRatAnti-MouseTer119 is 20 μg / ml, and incubate at 6°C for 10 minutes;

[0042] 5. Add 0.1ml PBS, flow cytometry analysis, thiazole orange excitation light 488nm, emission light 530nm, APC excitation light 638nm, emission light 660nm;

[0043] 6. Collect 20,000 cells, and use flow cytometry software to analyze the cell distribution of fluorescent populations.

Embodiment 3

[0045] 1. Take BALB / c mouse bone marrow cells, count, and take 1 million cells;

[0046] 2. Add 2ml of thiazole orange staining solution to the mouse bone marrow cells, the concentration of the thiazole orange staining solution is 0.05 μg / ml, incubate at room temperature for 60 minutes, wash, centrifuge, and discard the supernatant;

[0047]3. Add 0.15ml rat IgG for blocking, the concentration of the rat IgG is 0.1mg / ml, incubate at 4°C for 10min;

[0048] 4. Add 0.15ml APCRatAnti-MouseTer119, the concentration of APCRatAnti-MouseTer119 is 1 μg / ml, and incubate at 10°C for 60min;

[0049] 5. Add 0.1ml PBS, flow cytometry analysis, thiazole orange excitation light 488nm, emission light 530nm, APC excitation light 638nm, emission light 660nm;

[0050] 6. Collect 20,000 cells, and use flow cytometry software to analyze the cell distribution of fluorescent populations.

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Abstract

The invention discloses a method for analyzing and evaluating the erythropoiesis function of mouse marrow through a flow cytometry. According to the method, mouse marrow cells are subjected to dyeing incubation with thiazole orange, rat IgG and APC Rat Anti-mouse Ter119. Incubation sequence is that firstly, the mouse marrow cells and thiazole orange are incubated for appropriate time, centrifugation and supernatant abandoning are conducted, then, rat IgG is added to be incubated for appropriate time, and APC Rat Anti-mouse Ter119 is added to be incubated for appropriate time. Finally, PBS is added, the proportions of mature erythrocytes, reticulocyte, normoblast and proerythroblast in all cells of the mouse marrow are quantitatively analyzed on the flow cytometry, and therefore the erythropoiesis function of the mouse marrow is analyzed and evaluated. The method is quick, accurate, simple, easy to practice, low in cost and beneficial to application and popularization.

Description

technical field [0001] The invention relates to the technical field of flow cytometry, in particular to a method for analyzing and evaluating mouse bone marrow erythropoietic function by using flow cytometry. Background technique [0002] Mature erythrocytes are the most abundant type of blood cells in the body's blood, and are the most important medium for the body to transport oxygen through the blood. In recent years, studies have found that erythrocytes are also important immune cells of the body, and play an important role in fighting diseases and tumors. Mature erythrocytes are derived from erythroid progenitor cells in the bone marrow erythroid system through the gradual differentiation and development of proerythrocytes, immature erythrocytes and reticulocytes. Therefore, detecting the proportion of red blood cells in each period to analyze and evaluate the bone marrow erythropoietic function is of great significance for further exploring the role of red blood cells ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
CPCG01N33/53
Inventor 赵云雪荆卫强金星李秀秀
Owner 江苏健易达医疗科技有限公司
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