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Single-cell protein detecting method for flow type combination ICP-MS on basis of metal isotope labels

A technology of isotope labeling and single-cell protein, which is applied in the direction of measuring devices, biological testing, material inspection products, etc., can solve the problems of ICP-MS such as high price, large interference, and few detection channels, so as to save specimens and reagents, and solve the problem of fluorescence Cross-color, avoid the effect of compensation calculation

Inactive Publication Date: 2017-10-17
马鞍山普梅森细胞检测技术有限公司
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  • Abstract
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Problems solved by technology

[0010] Aiming at the problems of few detection channels of existing flow cytometers, mutual interference between signals, complex and time-consuming detection of multiple parameters, and problems such as expensive ICP-MS, large interference, and inability to realize single-cell level detection, the present invention provides A single-cell protein detection method based on metal isotope labeling flow cytometry combined with ICP-MS, which uses metal element labeling to label antibody dyes, uses mass spectrometry to perform multi-parameter quantitative detection of single cells, and is applied to single-cell proteomics analysis

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  • Single-cell protein detecting method for flow type combination ICP-MS on basis of metal isotope labels
  • Single-cell protein detecting method for flow type combination ICP-MS on basis of metal isotope labels
  • Single-cell protein detecting method for flow type combination ICP-MS on basis of metal isotope labels

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Embodiment 1

[0047] attached Figure 5 It is a comparison of the schematic diagrams of the traditional flow cytometer (upper panel) and this implementation (lower panel). Traditional flow cytometry uses fluorescent dyes to label antibodies, and then the antibodies bind to the antigens, surrounded by sheath fluid, and pass through the flow chamber to form a single-cell suspension. After passing through the laser, the forward scattering angle, side scattering angle, fluorescence The signal light information is transmitted to the computer for analysis via the PMT. In this implementation, metal element labels are used instead of fluorescent dye labels, and a single-cell suspension is formed through a nebulizer, and the single-cell suspension is ionized by a plasma torch, and detected by TOF. Due to the use of the technology of the present invention, the data volume and data dimension of the single-cell protein level detection results have increased sharply, so when analyzing data, various dim...

Embodiment 2

[0063] The steps of the sample pretreatment method based on flow cytometry combined with ICP-MS single-cell protein detection include the following:

[0064] (1) Processing and cryopreservation of human peripheral blood samples (extraction of mononuclear cells-cell viability staining-cell stimulation-cell fixation)

[0065] 1. Reagent preparation. a. Reagent equilibrated to normal temperature: Ficoll lymphocyte separation medium; Ca 2+ / Mg 2+ Free PBS; 2×Fixation Solution (3.2% PFA in PBS); Human red blood cell lysate (optional); Cell Staining Buffer (0.5% BSA+0.02% NaN 3 in PBS). b. Reagents that need to be preheated: FBS Free DMEM / 1640 (37°C). c. Place the following reagents on ice: Cell Staining Buffer containing 10% DMSO; Cell Staining Buffer; Cisplatin 5mM;

[0066] 2. Collection and transportation of whole blood samples. Blood sample collection is an important part of quality control before analysis. It is recommended to use heparin or citrate as an anticoagulant....

Embodiment 3

[0078] A single-cell protein detection method based on metal isotope labeling flow cytometry combined with ICP-MS, the steps are:

[0079] (1) Sample labeling: according to the aforementioned method, specific antibodies labeled with metal elements are used as the reporter of the target expression level; the metal elements used for labeling have an atomic mass between 88-210Da, and the metals commonly used for labeling The element is a lanthanide metal element. In this embodiment, the protein CD8a with an Nd isotope mass of 146 and the protein CD4 with an Nd isotope mass of 145 were respectively used.

[0080] (2) Atomization treatment: Use a peristaltic pump to evenly send the marked sample solution into the atomizer for atomization treatment to remove moisture; the atomization temperature is about 200 °C, and the liquid flow channel in the atomizer is Argon environment, the flow rate of argon is 0.15L / min.

[0081] (3) Plasma ionization: single-cell atomized hanging droplets...

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Abstract

The invention discloses a single-cell protein detection method based on metal isotope labeling flow cytometry combined with ICP-MS, and belongs to the technical field of flow cytometry single-cell protein detection. The present invention uses metal element tags to mark antibodies or dyes, which fundamentally solves the problem of fluorescent cross-color, uses the principle of mass spectrometry to perform multi-parameter quantitative detection of single cells, realizes the simultaneous measurement of dozens of parameters, and is applied to single-cell proteomics academic analysis. The fusion of the two experimental platforms of mass spectrometry and flow cytometry not only inherits the high-speed analysis characteristics of traditional flow cytometry, but also has the high-resolution capability of mass spectrometry detection. The metal tag elements that can be used in the present invention are rich in types, and the content in cells is extremely low. At the same time, metal elements are covalently coupled to antibodies through polychelates. The non-specific binding capacity of the product is extremely low, so the background of the signal is extremely low.

Description

technical field [0001] The invention belongs to the technical field of single-cell protein detection by flow cytometry, in particular, it relates to a protein labeling technology, and more specifically, it relates to a single-cell protein detection method based on metal isotope labeling flow cytometry combined with ICP-MS. It involves the simultaneous detection of at least 37 kinds of protein expression on a single-cell scale. Background technique [0002] Flow cytometry is a method that uses a laser beam to excite cells that flow in a single line, and detects its scattered light and fluorescence, so as to complete the one-by-one, multi-parameter, and rapid analysis of single-line cells or biological particles in a fast linear flow state. Qualitative and quantitative analysis or sorting technology has the characteristics of fast detection speed, many measurement parameters, large amount of collected data, comprehensive analysis, high sorting purity, and flexible methods. It ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/58G01N15/14
CPCG01N33/6848G01N15/14G01N33/58
Inventor 丁显廷张婷李轶洋
Owner 马鞍山普梅森细胞检测技术有限公司
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