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Preparation and detection methods of an animal heart single-cell suspension

A single-cell suspension and detection method technology, which is applied in the field of preparation and detection of animal heart single-cell suspension, can solve the problems of complicated operation of the detection method and no preparation method of cardiac single-cell suspension, etc. Reliable digestion, easy to use results

Inactive Publication Date: 2018-03-02
SHANGHAI PUTUO DISTRICT CENT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a preparation and detection method of animal cardiac single-cell suspension, which solves the problems that the prior art does not provide a specific cardiac single-cell suspension preparation method, and the detection method is complicated to operate, and can be stable, Reliable preparation of cardiac single-cell suspensions with simple detection methods, enabling comprehensive analysis of cell subpopulations (cell populations with different cell antigens) in cardiac single-cell suspensions

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0028] A preparation method of animal heart single cell suspension, the method comprising:

[0029] Step 1: making an animal model of cardiac ischemia-reperfusion;

[0030] Step 2: Take isolated animal heart tissue, rinse to remove blood, add cardiac digestive juice, cut into pieces, mix and digest; chopping is easy to operate, and can expand the contact area between myocardial tissue and digestive juice;

[0031] Step 3: filter the mixed solution obtained in step 2, centrifuge, remove the supernatant, stop the digestion, add phosphate buffer for suspension, add red blood cell lysate, centrifuge, remove the supernatant, add phosphate buffer for suspension, and obtain the animal heart single cell suspension.

[0032] In step 2, the heart digestion solution contains: RPMI-1640 medium (purchased from Hyclone, model SH30809.01B), Collagenase I (collagenase I, purchased from Gibco, model 17100017) and DNase (deoxyribonuclease, deoxyribonuclease ). The DNase is DNase I (purchased...

experiment example 1

[0047] Experimental Example 1 Making an Animal Cardiac Ischemia-Reperfusion Model

[0048] The preparation method of the animal heart ischemia-reperfusion model is as follows:

[0049] Step 1': Ether anesthetize, mouse endotracheal intubation, connect ventilator, set ventilator tidal volume 200-300μL, frequency 120 beats / min, connect electrocardiogram;

[0050] Step 2': Incision of the left anterior thorax, separating the subcutaneous tissue layer by layer, exposing the surgical field with a thoracic spreader in the third intercostal space, opening the pericardium, exposing the heart, looking for the anterior descending branch with the left atrial appendage and conus pulmonary artery as the mark, and using 8 -0 silk thread was inserted into the needle at 1-2mm below the left atrial appendage. In the sham operation group (positive control group), the thread was only threaded without ligation. In the ischemia-reperfusion operation group, a slipknot was tied after threading twice...

experiment example 2

[0054] Experimental example 2 Preparation of animal heart single cell suspension

[0055] Take the mouse heart at 24h, 48h, 72h, and 7d of reperfusion respectively, and prepare the cardiac single cell suspension. The specific operation is as follows:

[0056] (1) Take about 0.2 g of isolated animal heart tissue, rinse with PBS to remove blood, add 1 mL of heart digestion solution to a 2 mL centrifuge tube, transfer to a 15 mL centrifuge tube, add 10 mL of heart digestion solution, and place at room temperature for 60 minutes, mixing intermittently. Uniformly, a mixed solution is obtained;

[0057] (2) Filter the mixed solution obtained above with a 140-mesh filter, collect the filtrate in a 15mL centrifuge tube, centrifuge at 1100rpm for 10 minutes, remove the supernatant, add 200uL PBS to suspend, add 2mL red blood cell lysate, After 5 minutes in a dark room, centrifuge at 1100 rpm for 5 minutes, remove the supernatant, add 1 mL of phosphate buffer to suspend, and obtain ani...

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Abstract

Preparation and detection methods of an animal heart single-cell suspension are disclosed. The preparation method includes 1) preparing an animal heart ischemia reperfusion model; 2) preparing an animal isolated hear tissue, flushing the tissue to remove blood, adding heart digestive juice, cutting the tissue into pieces, fully mixing the tissue pieces with the digestive juice and performing digestion; and 3) filtering a mixture solution obtained in the step 2), performing centrifugation, removing a supernatant liquid, terminating digestion, adding a phosphate buffer liquid for suspension, adding an erythrocyte lysis solution, performing centrifugation, removing a supernatant liquid, and adding a phosphate buffer liquid for suspension to obtain the animal heart single-cell suspension. Thepreparation method can reduce destroy to cells, and the single-cell suspension preparation method which is reliable and stable is provided. The number of cells in the prepared suspension is high. Thedetection method is simple to operate and comprehensive in data.

Description

technical field [0001] The invention relates to a preparation method of a heart cell suspension, in particular to a preparation and detection method of an animal heart single cell suspension. Background technique [0002] Mouse animal model experiments are an important method for scientific research. Compared with animal models such as rats, the classification of inflammatory cells, cell markers, and experimental-related antibodies derived from mice are more detailed and abundant in experiments on inflammation and immunity. The study of inflammatory cells requires not only qualitative and morphological studies, but quantitative and functional studies can provide more accurate and comprehensive information. [0003] In the animal model of mouse cardiac ischemic injury, the lesions are focally distributed, and the pathological sections are easily disturbed by sampling errors and the observation field of view. [0004] With the continuous development of heart disease research,...

Claims

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Application Information

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IPC IPC(8): G01N15/14G01N1/28
CPCG01N1/28G01N15/14
Inventor 刘鸣
Owner SHANGHAI PUTUO DISTRICT CENT HOSPITAL
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