Method for detecting antigen expression level by using flow cytometry
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A flow cytometer and cell technology, applied in the field of flow cytometry, can solve the problem of inability to accurately detect weak antigen expression and other problems
Inactive Publication Date: 2013-03-27
JIANGSU PROVINCE HOSPITAL
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[0005] Aiming at the problem that flow cytometry cannot accurately detect weak antigen expression in the above-...
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Embodiment 1
[0020] Example 1: Detection of ZAP-70 expression level in chronic lymphocytic leukemia tumor cells
[0021] 1. Preparation of mononuclear cells: freshly submitted chronic lymphocytic leukemia peripheral blood samples 2ml, lymphocyte separation medium to separate mononuclear cells, adjust the concentration of mononuclear cell suspension to ±10 6 cells / 100 μl.
[0022] The above-mentioned target cells can be prepared by selecting different existing technologies according to different samples in actual situations, and the samples can be bone marrow, peripheral blood or lymph nodes.
[0023] 2. Immunofluorescence staining of cells.
[0024] 2.1 Add 100 μl of the prepared single cell suspension to each tube, a total of two tubes. Add 20 μl of CD3+CD5-FITC / CD19-PerCP mouse anti-human monoclonal antibody (from BD Pharmingen, USA) to both tubes, vortex and mix well, and react at room temperature for 30 minutes in the dark.
[0025] 2.2 Wash once with PBS, 1000 rpm, centrifuge to re...
Embodiment 2
[0041] Example 2: Detecting the expression level of CD137L in acute leukemia leukemia tumor cells
[0042] 1. Preparation of mononuclear cells: 2ml of freshly drawn acute leukemia bone marrow specimen, separate mononuclear cells with lymphocyte separation medium, adjust the concentration of mononuclear cell suspension to ±10 6 cells / 100 μl.
[0043] The above-mentioned target cells can be prepared by selecting different existing technologies according to different samples in actual situations, and the samples can be bone marrow, peripheral blood or lymph nodes, etc.
[0044] 2. Immunofluorescence staining of cells.
[0045] 2.1 Add 100 μl of the prepared single cell suspension to two tubes in total. Add 20 μl each of CD137L-PE / CD45-PerCP5.5 mouse anti-human monoclonal antibody (from BD Pharmingen, USA) to the test tube, 20 μl each of IgG1-PE / CD45-PerCP5.5 in the control tube, vortex and mix well, and keep away from room temperature. Light reaction for 30 minutes.
[0046] ...
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Abstract
The invention discloses a method for detecting an antigen expression level by using a flow cytometry. The method comprises the steps of firstly, carrying out immunofluorescent staining on a target cell, then detecting the stained cell by adopting the flow cytometry and carrying out data calculation and analysis, wherein when the parameter of the low cytometry is set, the resolution of a fluorescence channel to be detected is reduced; and obtaining a geometric average fluorescence intensity of an isotype control or a negative cell and a target cell to-be-detected antigen, figuring out respective geometric average fluorescence intensity rates with the geometric average fluorescence intensity rate of the isotype control or negative cell as a critical value, and if the geometric average fluorescence intensity rate of the target cell to-be-detected antigen is higher than that of the isotype control or negative cell by a preset value, the expression of the target cell to-be-detected antigen is achieved. According to the invention, the accuracy and the precision of target cell determination are effectively increased, experimental errors and subjective judgment errors of weak antigen expression are maximally reduced, and the flow cytometry is effectively promoted to be applied to multiple fields.
Description
technical field [0001] The invention relates to the technical field of flow cytometry, in particular to a flow cytometry analysis method for detecting the expression level of weak antigens by using a flow cytometer. Background technique [0002] Flow cytometry (Flow Cytometry, FCM) is a detection method for quantitative analysis and sorting of single cells or other biological particles at the functional level. It uses laser as the excitation light source, can analyze tens of thousands of cells at high speed, and can measure multiple parameters from one cell at the same time. It has the advantages of fast speed, high precision, and good accuracy. It has become the most advanced cell quantitative analysis technology , is widely used in clinical medicine and various biological research fields. At present, most flow cytometers believe that they can detect weak antigens with as low as 100 fluorescent molecules on a single cell in terms of theoretical performance parameters. How...
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