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Method for imaging live nucleus and cytoplasm and application thereof in monitoring live nucleus and cytoplasm signal pathway

A technology of living cell nuclei and living cells, applied in biological testing, material inspection products, fluorescence/phosphorescence, etc., can solve the problems of cumbersome operation of optical path changes, difficulty in cell line selection and maintenance, and impossibility of dynamic research

Inactive Publication Date: 2010-06-16
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, they bring corresponding methodological deficiencies: the premise of the implementation of method (1) is to screen for cell lines with high expression of fluorescent proteins, and this abnormally high expression will have a potential impact on the normal physiological activities of cells on the one hand, and on the other hand On the one hand, it also brings difficulties to the selection and maintenance of cell lines
Method (2) adds a nuclear fluorescent staining procedure in the sample preparation process, the entry of exogenous fluorescent dyes may have a certain impact on the physiological state of the cells, and if used in the case of low expression cell lines ( It is difficult to collect experimental data under the smallest possible external interference to the cells, so that it is impossible to realize the dynamic research under the state of causing the smallest possible external interference to the cells; method (3) frequent optical path changes bring cumbersome operations

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  • Method for imaging live nucleus and cytoplasm and application thereof in monitoring live nucleus and cytoplasm signal pathway
  • Method for imaging live nucleus and cytoplasm and application thereof in monitoring live nucleus and cytoplasm signal pathway
  • Method for imaging live nucleus and cytoplasm and application thereof in monitoring live nucleus and cytoplasm signal pathway

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Embodiment 1

[0053] The first embodiment of the present invention is to use the method of the present invention to observe the translocation process of NF-κB marked by gene recombination technology GFP between nucleoplasm and tumor necrosis factor (Tumor necrosis factor) in human lung adenocarcinoma cells (ASTC-a-1). NecrosisFactor, TNF) as an activator of NF-kB signaling pathway.

[0054] Culture of human lung adenocarcinoma cell (ASTC-a-1): cell culture medium is RPMI 1640, add 15% fetal bovine serum, 2mmol / L glutamine (Glutamine), 25mmol / L HEPES, 100IU / ml penicillin and 100mg / mL streptomycin. For cell transfection, 30%-50% confluent human lung adenocarcinoma cells were co-cultured with a suspension of Lipofectin reagent and a sufficient amount of recombinant plasmid vector (gifted by Professor Schmid, University of Vienna) for 6 hours, and then supplemented with an appropriate amount of fresh culture medium for expansion. Increase culture. The process of transfection is the process o...

Embodiment 2

[0058] The second embodiment of the present invention is to use the present invention to observe the translocation process of STAT marked by the gene recombination technology GFP between nucleoplasm in human lymphocytes - Interferon (Interferon) is used as the activator of the signaling pathway STAT.

[0059] Lymphocytes were used as host cell lines for validation of the JAK / STAT signaling pathway. Whole blood comes from healthy blood donors at the blood bank, and lymphocytes are obtained from heparin-anticoagulated blood separated by lymphocyte separation fluid. RPMI1640 was used as the lymphocyte culture medium, and 10% fetal bovine serum, HEPES, antibiotics, interleukin-2 (IL-2), and phytohemagglutinin (PHA) were added. Construction of the transfection vector: the genes of the STATs family (STAT 1-6) were cloned into the carboxyl terminus of the plasmid vector pEGFP-C1 (Catalog #6084-1, Clontech). Lipofectin reagent and a sufficient amount of recombinant plasmid vectors we...

Embodiment 3

[0063] Culture of human skeletal muscle cells: cell culture medium is DMEM, add 10% fetal bovine serum, 5uM growth factor (GrowthFactor), 2mM glutamine (Glutamine), 25mmol / L HEPES, 100IU / ml penicillin and 100mg / mL streptavidin white. For cell transfection, 30% to 50% confluent human skeletal muscle cells were co-cultured with a suspension of Lipofectin reagent and a sufficient amount of recombinant plasmid vector for 6 hours, and then an appropriate amount of fresh culture medium was supplemented for expansion culture. Construction of the transfection plasmid: the androgen receptor gene was inserted into the amino terminal of the commercialized EGFP plasmid vector, the green fluorescent protein (GFP) gene was recombined with the androgen receptor gene, and the combined gene (plasmid vector) was transfected Transfection into skeletal muscle cells, the expression product of this foreign gene in skeletal muscle cells is the fusion protein of androgen receptor and GFP. When using...

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Abstract

This invention discloses a method for imaging live nucleus and cytoplasm, which comprises the following steps that: (a) live cells are irradiated by a near-infrared femtosecond laser which is focused by a microscope; (b) the live cells produce two-photon fluorescence and second harmonic under the action of the near-infrared femtosecond laser focused by the microscope; and (c) the two-photon fluorescence and the second harmonic are subjected to microscopic imaging at the same time, the two-photon fluorescence performs microscopic imaging on target molecules which are marked by fluorescence, and the second harmonic performs microscopic imaging on the nucleus. The invention also discloses application of the method for imaging the live nucleus and cytoplasm in monitoring a live nucleus and cytoplasm signal pathway, eliminates photoinduced toxicity and photobleaching, and can observe the live cells for a long time without affecting the activity of the cells and furthest maintain the functional environment of the cells. The method can be applied to the level layer research of cell molecules in a tumor generation mechanism and the research of a pharmacodynamical mechanism of traditional Chinese medicinal herbs.

Description

technical field [0001] The invention relates to the fields of cell molecular biology and nonlinear optical microscopic imaging, in particular to a living cell nucleoplasmic imaging method and its application in the monitoring of living cell nucleoplasmic signal transduction pathways. Background technique [0002] Multicellular organisms are a busy and orderly cell society. The maintenance of this kind of society not only depends on the material metabolism and energy metabolism of cells, but also depends on cell communication and signal transmission, so as to coordinate their behavior in different ways, such as Cell growth, division, death, differentiation and their various physiological functions. [0003] The information transduction between organism cells can be realized through the direct contact of adjacent cells, but more importantly and more commonly, cells secrete various chemical substances to regulate the metabolism and function of themselves and other cells. Cells...

Claims

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Application Information

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IPC IPC(8): G01N33/48G01N21/64
Inventor 邓小元金鹰
Owner SOUTH CHINA NORMAL UNIVERSITY
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