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Sample pretreatment method based on flow cytometry combined with icp-ms single-cell protein detection

An ICP-MS, single-cell protein technology, which is applied in the field of pretreatment of human peripheral blood samples, can solve the problem that the pretreatment method cannot be applied to the detection of metal label-labeled cells, and achieves overcoming easy quenching, convenient processing, and non-specific background. high effect

Active Publication Date: 2020-07-31
马鞍山普梅森医学检验实验室有限公司
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Problems solved by technology

[0017] Aiming at the problem that the existing flow cytometry sample pretreatment method cannot be applied to the detection of metal tagged cells, the present invention provides a sample pretreatment method based on flow cytometry combined with ICP-MS single-cell protein detection, which can label metal element tags The antibody binds to the cell surface antigen, and the labeled cells are mixed with the beads used as an internal reference for standardization. During the pretreatment process, dead cells and living cells are distinguished to avoid the excessive number of dead cells affecting the analysis and interpretation of the experimental results. Single cells and cell dimers, trimers and even multimers can well meet the needs of flow cytometry combined with ICP-MS single cell protein detection

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  • Sample pretreatment method based on flow cytometry combined with icp-ms single-cell protein detection

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Embodiment 1

[0046] like figure 1 As shown, the steps of the sample preparation method based on flow cytometry combined with ICP-MS single-cell protein detection include the following:

[0047] (1) Processing and cryopreservation of human peripheral blood samples (extraction of mononuclear cells - cell viability staining - cell stimulation - cell fixation)

[0048] 1. Reagent preparation. a. Reagents are equilibrated to room temperature: Ficoll lymphocyte separation solution; Ca2+ / Mg 2+ Free PBS; 2×Fixation Solution (3.2% PFA in PBS); Human erythrocyte lysate (optional); Cell Staining Buffer (0.5% BSA+0.02% NaN) 3 in PBS). b. Reagents requiring preheating: FBS Free DMEM / 1640 (37°C). c. Place the following reagents on ice: Cell Staining Buffer containing 10% DMSO; Cell Staining Buffer; Cisplatin 5mM;

[0049] 2. Collection and transportation of whole blood samples. Blood sample collection is an important part of pre-analytical quality control. It is recommended to use heparin or cit...

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Abstract

The invention discloses a sample pretreatment method based on flow combination ICP-MS (Inductively Coupled Plasma Mass Spectrometry) single cell protein detection, and belongs to the technical field of sample pretreatment of flow cytometry. The sample pretreatment method comprises the following steps: (1) collecting and transporting a whole blood sample; (2) performing PBMC (Peripheral Blood Mononuclear Cell) cell separation; (3) performing cell active dyeing; (4) performing cell stimulation; (5) performing cell immobilization; (6) performing surface antibody dyeing; (7) performing intracellular phosphorylated protein dyeing; (8) performing single cell labeling, and the like. With the combination of a metal element labeled antibody with cell surface antigen, labeled cells are mixed with beads as internal reference for standardization, dead cells and living cells are distinguished in the pretreatment process, then the situation that the testing result analysis and description are affected by too many dead cells is avoided, single cells are distinguished from cell dimmers, cell trimers or even cell multimers, and flow combination ICP-MS single cell protein detection requirements can be very well met.

Description

technical field [0001] The invention belongs to the technical field of sample pretreatment of flow cytometry, and more particularly relates to a sample pretreatment method based on flow cytometry combined with ICP-MS single cell protein detection, in particular to a pretreatment method of human peripheral blood samples. Background technique [0002] Flow cytometry is a method that uses a laser beam to excite cells flowing in a single row, and detects its scattered light and the fluorescence carried by it, so as to complete the one-by-one, multi-parameter, rapid analysis of a single row of 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, flexible methods, etc. It is widely used in clinical medicine, cytology, biology, Microbiology, pharmacy, reproductive ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N15/14G01N27/62
CPCG01N15/14G01N27/62G01N2015/1486
Inventor 丁显廷张婷李轶洋
Owner 马鞍山普梅森医学检验实验室有限公司
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