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Method for flow cytometry analysis of stem cell cytokines

A technology of flow cytometry and cytokines, which is applied in the field of flow cytometry, can solve the problems of high chip cost, time-consuming, and low throughput of microfluidic single-cell proteomics technology, and achieve low cost, low preparation operation, and easy operation. easy effect

Active Publication Date: 2021-05-14
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the throughput of microfluidic single-cell proteomics technology is still low, and usually only a few thousand cells can be detected at a time; microfluidic single-cell proteomics technology requires high chip preparation costs; It is cumbersome, requires certain skills of the operator, and is more time-consuming

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  • Method for flow cytometry analysis of stem cell cytokines
  • Method for flow cytometry analysis of stem cell cytokines
  • Method for flow cytometry analysis of stem cell cytokines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Flow cytometric analysis of cytokines in stem cells of the LPS+Pam3Csk4 stimulated cell model

[0068] 1. Experimental method

[0069] (1) Mouse hematopoietic stem cells (Lin low / - Sca1 + c-Kit high LSK cells) culture

[0070] Under the infection model, cells are stimulated with LPS+Pam3Csk4, and the culture system of this model is:

[0071] (1) Mouse hematopoietic stem cells (Lin low / - Sca1 + c-Kit high LSK cells) were cultured in SFEM medium, stimulated with LPS+Pam3Csk4, added SCF (stem cell growth factor PEPEOTECH, product number: 250-0350ng / mL) according to the working concentration and cultured for 12 hours;

[0072] (2) The cells are still under the stimulation of LPS+PamCsk43, and the cells are stimulated with an activation stimulator to accelerate protein synthesis and store cytokines in the cells;

[0073] (3) After 6 hours of stimulation, the membrane was fixed and ruptured to detect the expression level of cytokines in the cells by flow c...

Embodiment 2

[0091] Example 2 Flow cytometric analysis of tumor-bearing mice spleen stromal supernatant induced hematopoietic stem cells stress cytokines of hematopoietic stem cells

[0092] 1. Experimental method

[0093] (1) Mouse hematopoietic stem cells (Lin low / - Sca1 + c-Kit high LSK cells) culture

[0094] The training system for this model is:

[0095] (1) Mouse hematopoietic stem cells (Lin low / - Sca1 + c-Kit high LSK cells) were cultured in SFEM medium, the cells were stimulated with the spleen stroma supernatant of tumor-bearing mice, and SCF (stem cell growth factor) was added according to the working concentration to cultivate for 4 days;

[0096] (2) Still under the stimulation of the spleen stroma supernatant, the cells were stimulated with an activation stimulator to accelerate protein synthesis and store cytokines in the cells (see Table 1 of Example 1 for the dosage of the activation stimulator).

[0097] (3) After 6 hours of stimulation, the membrane was fixed ...

Embodiment 3

[0102] Example 3 Medium and cytokine flow cytometric analysis on cytokines of stem cells stimulated by LPS+Pam3Csk4 cell model

[0103] 1. Experimental method

[0104] According to the experimental method (1) of Example 1 (1) to treat (in SFEM medium, stimulate the cells with LPS+Pam3Csk4, add SCF according to the working concentration and culture for 12 hours), and then transfer the cells to the medium: IMDM In +10% FBS, one or more of the cytokines SCF, TPO and Flt3L were added according to the working concentration, and an activation stimulator was added to stimulate for 6 hours (see Table 1 of Example 1 for the dosage of the activation stimulator).

[0105] The dosage of table 4 cytokines:

[0106] Cytokines Cytokine source working concentration Stem Cell Factor (SCF) PEPEOTECH, Item No.: 250-03 50ng / mL TPO PEPEOTECH, Item No.: 315-14 20ng / mL FLT3 PEPEOTECH, Item No.: 250-31L 100ng / mL

[0107] Then follow the method of Example...

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Abstract

The invention discloses a method for flow cytometry analysis of stem cell cytokines, and provides a staining technology capable of truly simulating in-vivo conditions of stress hematopoiesis of hematopoietic stem cells through proper culture and stimulation conditions and accurately detecting the expression condition of the stem cell cytokines in the hematopoietic stem cells through a fixed membrane rupture flow cytometry analysis technique. The method is simple and convenient to operate and low in requirements on cell suspension preparation operation, and the stress hematopoietic stem cells conforming to the real condition of in-vivo infection can be obtained only by skillfully mastering a cell culture technology and conducting culturing according to the conditions designed by the technology. Moreover, operations such as chip preparation are not needed, so cost is relatively low. Extracellular cytokines secreted by a compound are retained in cells, and intracellular and extracellular cytokine expression conditions of the cells can be effectively and accurately measured. A technical foundation is laid for further deeply discussing a regulation mechanism of cytokine expression and secretion of hematopoietic stem precursor cells.

Description

technical field [0001] The invention relates to the technical field of flow cytometry, in particular to a method for cell flow cytometry analysis of stem cell cytokines. Background technique [0002] Flow cytometry is a powerful technique with broad and in-depth applications in immunology, molecular biology, bacteriology, virology, tumor biology, and infectious disease monitoring to study cell populations with high precision. Flow cytometry enables the simultaneous analysis of multiple parameters of a single cell, such as: expression of cell surface and intracellular molecules, identification and determination of different cell types in a heterogeneous cell population, assessment of purity of isolated subpopulations, and analysis of cell size and volume Wait. [0003] Flow cytometry is a technique that can rapidly analyze single cells or particles encapsulated in buffered saline and flowed through single or multiple lasers with corresponding visible light scattering and flu...

Claims

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Application Information

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IPC IPC(8): C12N5/0789G01N33/569
CPCC12N5/0647G01N33/56966C12N2501/125C12N2501/145C12N2501/26C12N2501/999G01N2469/10
Inventor 郑利民罗舒凤吴翀
Owner SUN YAT SEN UNIV
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