Intracellular amino acid metabolic profiling analysis method

A metabolic profile and analysis method technology, applied in the field of high-throughput analysis of intracellular amino acid metabolic profile, can solve problems such as difficulty in obtaining metabolome data, achieve high throughput of cell culture and operation, avoid the influence of intracellular metabolism, and optimize Effect of extracting repeatability and recovery

Inactive Publication Date: 2016-04-27
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, in the study of stem cells, the number of stem cells that can be used for metabolic profiling is relatively small, and conventional analysis strategies are difficult to obtain rich metabolome data

Method used

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  • Intracellular amino acid metabolic profiling analysis method
  • Intracellular amino acid metabolic profiling analysis method
  • Intracellular amino acid metabolic profiling analysis method

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Experimental program
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Effect test

Embodiment 1

[0026] Investigation on the analysis method of intracellular amino acid metabolism profiling in a small number of cells:

[0027] 1) Inoculate and culture HepG2 cells on a 96-well plate

[0028] at 175cm 2 HepG2 cells were adherently cultured in cell culture flasks: DMEM high-glucose medium with 10% FBS was given, and the cells were grown to 60-70% confluence at 37°C and 5% CO2 under constant conditions.

[0029] Cells were trypsinized and resuspended in DMEM medium containing 10% FBS. For cell counting, prepare a density of 2 x 10 4 cells / ml of cell suspension was used to inoculate 96-well plates.

[0030]In a 96-well plate, 100 μL of the above-mentioned HepG2 cell suspension (the suspension was prepared with 10% FBS in DMEM medium) was inoculated in each well, and kept at 37° C. and 5% CO 2 for 48 hours.

[0031] 2) Cell quenching and intracellular metabolite extraction

[0032] After HepG2 cells grew for 48 hours, the cells growing in each well of the 96-well plate wer...

Embodiment 2

[0059] Profile analysis of intracellular amino acid metabolism in HepG2 cells with fatty acid intervention:

[0060] 1) HepG2 cell culture and fatty acid intervention

[0061] at 175cm 2 Adhesive culture of HepG2 cells in cell culture flasks: give 10% FBS in DMEM high-glucose medium, and grow the cells to 60-70% confluence at 37°C and 5% CO2 under constant conditions.

[0062] Cells were trypsinized and resuspended in DMEM with 10% FBS. For cell counting, prepare a density of 2 x 10 4 cells / ml of cell suspension was used to inoculate 96-well plates.

[0063]In a 96-well plate, 100 μL of the above-mentioned HepG2 cell suspension (the suspension was prepared with 10% FBS in DMEM medium) was inoculated in each well, and kept at 37° C. and 5% CO 2 for 24 hours.

[0064] After the cells grew for 24 hours, they were subjected to fatty acid intervention. The fatty acids applied were palmitic acid (PA, C16:0), palmitoleic acid (POA, C16:1) and combinations of both. The method of...

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Abstract

The invention discloses an intracellular metabolic profiling high-flux analysis method of a few of cells (with the number of 103-104). The method comprises simple and efficient cell quenching and metabolite extraction, metabolite derivatization and mass spectrometric detection. The intracellular metabolic profiling high-throughput analysis method has the characteristics of simpleness, fastness, high sensitivity and good repeatability and is suitable for high-throughput analysis of metabolome of cells cultured through a 96-well plate and cells with a low yield, wherein the analysis fields comprise drug large-scale screening, evaluation and stem cell research.

Description

technical field [0001] The invention relates to the field of analytical chemistry, and is a high-throughput analysis method for the intracellular amino acid metabolism profile of a small number of cells. Background technique [0002] Under the theoretical framework of systems biology, "multi-omics" research that links metabolic phenotypes and disturbances in metabolic pathways to their intrinsic pathophysiological mechanisms with the help of metabolic profiling has been increasingly used in cancer research. Stem cells and drug efficacy and drug toxicity research. Cell metabolic profiling is an important tool to achieve the above research purposes. In the analysis of cell metabolic profiling, time-consuming cell culture and complicated pretreatment steps have always been the main factors that limit the improvement of analysis throughput. A small number of cells (approximately 10 3 -10 4 ) rapid culture and high-throughput operation. In addition, in the study of stem cell...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
Inventor 许国旺周丽娜尹沛源高鹏路鑫
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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