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Method for digesting and extracting apoptosis rate in flow cytometry tissue by using digestion method

A technology of flow detection and digestion, which is applied in the field of basic medicine, can solve problems such as low reliability, poor accuracy, and large data influence, and achieve accurate and reliable results

Inactive Publication Date: 2013-07-24
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for tissues, especially the connective tissue with few cells and rich extracellular matrix such as human intervertebral disc nucleus pulposus, the commonly used method for detecting apoptosis is Tunel staining. This method has low sensitivity and poor accuracy, and eventually apoptotic cells The proportion needs to be counted and counted under the microscopic field of vision, the reliability is low, and the data is greatly affected by human influence

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0016] 1) Enzyme digestion of target tissue to collect cells

[0017] The collected tissues were carefully separated under sterile conditions and cut into 1×1mm 3 Fragments were digested with 0.25% trypsin at room temperature for 40 minutes, then centrifuged at a low speed (1000r / min) for 5 minutes, discarded the supernatant, digested with 0.025% type II collagenase at room temperature for 4 hours, and centrifuged at a low speed (1000r / min) after 5min, the supernatant was discarded, and PBS solution was added to disperse the cells by pipetting. Add the suspension to a centrifuge tube, centrifuge at 1000r / min for 5 minutes, suck out the supernatant, and then add PBS solution to blow the cells to obtain the digested target cells.

[0018] 2) Annexin V and PI double staining and FCM detection

[0019] The prepared single cell suspension (100 μl single cell suspension cell density about 1×10 6 Cells), using the Annexin V-FITC Apoptosis Detection Kit, the specific binding of Ann...

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Abstract

The invention provides a method for digesting and extracting apoptosis rate in flow cytometry tissue by using a digestion method. The method is as below: separating the collected tissue under sterile conditions; digesting the tissue in trypsin and centrifuging; discarding a supernatant; conducting static digestion at room temperature by using type II collagenase and centrifuging; discarding a supernatant; adding a PBS solution; blowing, beating and centrifuging the cells and adding the cells into a centrifuge tube; centrifuging and sucking out a supernatant; then adding the PBS solution, and blowing and beating the cells to obtain digested objective cells; preparing the objective digested cells into a solution with cell density of 1 * 10<6> per 100 muL single-cell suspension; using an Annexin V-FITC cell apoptosis detection kit to detect apoptosis through specific binding of an Annexin V antibody and phosphatidylserine. The method for extracting apoptosis rate in flow cytometry tissue by using digestion method provided by the invention provides an accurate, quantitative and reliable detection method for apoptosis related experiments. The results from the detection method are highly in accordance with project design, and are accurate and reliable.

Description

technical field [0001] The invention belongs to basic medicine and relates to a method for detecting cell apoptosis, in particular to a method for detecting cell apoptosis rate in tissue by digesting and extracting cells by flow cytometry. Background technique [0002] Apoptosis [Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer, 1972, 26:239–257.], also known as programmed Programmed cell death (PCD) occurs widely in various organs of the body. Cell apoptosis plays an important role in the aging, degeneration and renewal of tissues and organs. Current studies have found that apoptosis and human Alzheimer's disease [Anderson AJ, Su JH, Cotman CW. DNA damage and apoptosis in Alzheimer's disease: colocalization with c-Jun immunore activity, relationship to brain area, and effect of postmortem delay. J Neurosci,1996,16:1710–1719.],[Smale G,Nichols NR,Brady DR,Finch CE,Horton WE Jr.Evidence fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N1/28
Inventor 王海强刘志恒李新奎李昂高扬孙振陈宇飞张威林张泳照万中元
Owner FOURTH MILITARY MEDICAL UNIVERSITY