Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Mass spectrometric detection method and device for single organelle

A detection method and single-cell technology, applied in the field of organelle analysis, can solve the problems of inability to effectively detect and analyze the contents of organelles

Pending Publication Date: 2021-08-20
UNIV OF SCI & TECH OF CHINA
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the deficiencies of the prior art, the present invention provides a single cell organelle mass spectrometry detection method to solve the technical problem that the prior art cannot effectively detect and analyze the contents of the organelle

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Mass spectrometric detection method and device for single organelle
  • Mass spectrometric detection method and device for single organelle
  • Mass spectrometric detection method and device for single organelle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] This embodiment takes isolated HEK-293T cells as an example for introduction, and of course the present invention can also be used for mouse embryonic fibroblasts (MEF), mouse lung fibroblasts (MLF), bladder cancer cells (T24), human Immortalized bladder epithelial cells (SV-HUC-1), BxPC-3 cells, mouse cardiomyocytes and cardiac fibroblasts, mouse cerebral cortex neurons and glial cells, mouse peritoneal macrophages, mouse skin Cells such as fibroblasts (MEFs).

[0047] This embodiment discloses a single cell organelle mass spectrometry detection method, comprising the following steps:

[0048] Step 1. Cell culture

[0049] Culture of HEK-293T cells

[0050]HEK-293T cells used for passage were cultured in T25 culture flasks. Aspirate the culture medium in the culture bottle, add 1 mL of preheated trypsin to wash it again, add 1 mL of trypsin, and put it in a 37°C carbon dioxide incubator to digest for 2 minutes; add 4 mL of preheated culture medium to the culture bot...

Embodiment 2

[0098] The invention also discloses a way of freezing and recovering cells.

[0099] HEK-293T cell lines that can be subcultured are generally frozen in liquid nitrogen and resuscitated when used. In addition, cells with specific gene CRISPR / Cas9 knockouts are also cryopreserved and stored in liquid nitrogen. Cultured primary cells are generally not frozen.

[0100] Further, the invention also discloses a method for freezing and recovering cells.

[0101] HEK-293T cell lines that can be subcultured are generally frozen in liquid nitrogen and resuscitated when used. In addition, cells with specific gene CRISPR / Cas9 knockouts are also cryopreserved and stored in liquid nitrogen. Cultured primary cells are generally not frozen.

[0102] Cell Cryopreservation and Recovery

[0103] Among them, cryopreservation of HEK-293T cells includes the following steps:

[0104] 1) HEK-293T cells cultured in T75 culture flasks can be frozen when the density reaches 80%-90%.

[0105] 2) P...

Embodiment 3

[0115] In order to test the stability and reliability of the method of the present invention, the method of the present invention was used to investigate the transport of amino acids by the cationic amino acid transporter PQLC2 on the lysosome membrane.

[0116] PQLC2 can transport cationic amino acids in lysosomes to the cytoplasm, and the current generated by PQLC2-mediated cationic amino acid transport was recorded by whole lysosome patch clamp.

[0117] Such as Figure 3-8 As shown, HEK-293T cells overexpressing rPQLC2 compared with overexpressing EGFP-N1, recorded rPQLC2-mediated transport of cationic amino acids such as lysine, histidine and arginine to generate significant inward currents. Since the basic current generated by cationic amino acid transport mediated by the background level of PQLC2 is relatively small, there is no significant difference in the current recorded on the lysosomes of HEK-293T cells overexpressing EGFP-N1 and PQLC2 knockout.

[0118] Among th...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
lengthaaaaaaaaaa
diameteraaaaaaaaaa
lengthaaaaaaaaaa
Login to View More

Abstract

The invention discloses a mass spectrometric detection method for a single organelle. The method comprises a method for acquiring information of the single organelle by combining a patch clamp technology of the single organelle with a mass spectrometric technology. The invention further discloses a mass spectrum detection device for a single organelle. According to the method, a monolysosome patch clamp technology and an electrospray ionization source mass spectrometry technology are combined, so that a monolysosome mass spectrometry technology is established. The patch clamp technology can detect the activity of an ion channel or transporter on a lysosome membrane, and the electrospray ion source mass spectrometry technology can timely analyze a metabolic sample. After the electric signal of the lysosome is recorded by a lysosome patch clamp technology, the lysosome content is sampled for mass spectrometric detection, so that the components of the lysosome content are quantitatively analyzed, and the sample does not need to be pretreated. The method realizes simultaneous detection of lysosome functions and metabolic states. Compared with a traditional lysosome homogenate analysis method, the method has the advantage that the metabolic state of lysosome can be reflected more truthfully by a single lysosome mass spectrum technology.

Description

technical field [0001] The invention relates to the technical field of cell organelle analysis, in particular to a single cell organelle mass spectrometry detection method and a detection device. Background technique [0002] Lysosomes are single-membrane-enclosed vesicular organelles in eukaryotic cells that can degrade intracellular biological macromolecules. Lysosomes are heterogeneous, so it is necessary to study the metabolism of a single lysosome to further reveal the regulation of lysosome function. The prior art method for studying organelle metabolism mainly adopts the method of separating and purifying the organelles by differential centrifugation or density gradient centrifugation, and then homogenizing the organelles for component identification. This method exposes lysosomes to the non-cellular environment for a long time, which is easy to cause sample loss and the production of metabolic by-products. Kelly, B.M. et al. found that lysosomes are heterogeneous o...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/72
CPCG01N30/06G01N30/72
Inventor 熊伟朱洪影仓春蕾
Owner UNIV OF SCI & TECH OF CHINA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com