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Method for detecting protease-4 of human airway trypsin sample by using flow cytometry

A technology of flow cytometry and abnormal cells, which is applied in the field of detection of HATL4 by flow cytometry, can solve the problems of long detection cycle and large amount of bone marrow samples, and achieve the effect of short detection cycle, simple operation steps and pain relief

Active Publication Date: 2016-07-13
SUZHOU UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

At present, HATL4mRNA in the bone marrow cells of various leukemia patients can be detected by real-time fluorescent quantitative PCR (see "Expression of type II transmembrane serine protease HATL4 and the development of its monoclonal antibody" in Jiang Yizhi's master's thesis of Soochow University), but this method exists Disadvantages such as the large amount of bone marrow samples required and the long detection cycle

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  • Method for detecting protease-4 of human airway trypsin sample by using flow cytometry
  • Method for detecting protease-4 of human airway trypsin sample by using flow cytometry

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1: The CD45 / SSC dual-parameter scatter plot gating method is used to detect bone marrow samples.

[0021] In this example, 1 mL of bone marrow was used as a sample, and the puncture point was taken 1 cm behind and above the anterior superior iliac spine, and the sample was placed in a pre-heparinized 5 mL syringe for anticoagulation until use.

[0022] Aspirate 100 μL of anticoagulated bone marrow, put it into an experimental tube, add 10 μL each of CD45-PerCP, CD13-PE and HATL4-FITC and mix well, add different markers according to different abnormal cell groups: add CD13 to granulocytes, add Add CD14 to monocytes, add CD19 and CD3 to lymphocytes, and add isotype control to the control tube at the same time, then react at room temperature in the dark for 30 minutes, add 1 mL of hemolytic agent (add 8 g of NH to 1 L of water) 4 Cl, 1gKHCO 3 and 30mgEDTA-2Na, stirred evenly, and filtered with a 0.45μm microporous membrane to obtain), after mixing, hemolyzed in th...

Embodiment 2

[0025] Example 2: The CD45 / SSC dual-parameter scatter plot gating method is used to detect peripheral blood samples.

[0026] In this embodiment, 1 mL of peripheral blood is used as a sample, which is placed in a pre-heparinized 5 mL test tube for anticoagulation after sampling.

[0027] Draw 100 μL of anticoagulated peripheral blood, put it in a test tube, add 10 μL each of CD45-PerCP, CD13-PE and HATL4-FITC and mix well, add different markers according to different abnormal cell groups: add CD13 to granulocytes, Add CD14 to monocytes, add CD19 and CD3 to lymphocytes, and add isotype control to the control tube at the same time, then react at room temperature in the dark for 30 minutes, add 1 mL hemolytic agent (preparation method is the same as above), mix well, and then hemolyze in the dark for 15 minutes , after complete hemolysis, centrifuge at 2000rpm for 5min, discard the supernatant, wash once with PBS, centrifuge at 2000rpm for 5min, and wait for the test.

[0028] T...

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Abstract

The invention discloses a method for detecting protease-4 of a human airway trypsin sample by using flow cytometry. Specifically, the method comprises the following steps: (1) collecting a marrow or peripheral blood sample; (2) performing immunofluorescent labeling; (3) performing detection with a becton dickinson caliber. According to the detection method disclosed by the invention, a few of marrow cells canbe used as samples, and peripheralblood can be also used as a sample, so that pain of a patient in a sample collecting process can begreatly relieved, and the compliance of the patient is improved. Furthermore, the operation steps in the detection method disclosed by the invention are simple and convenient, and the detection period is short; the detection can be completed within only several hours; the detection period is greatly shortened, the progress of relevant researches can be accelerated favorably, and the time cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a method for detecting human airway trypsin-like protease 4 (HATL4), in particular to a method for detecting HATL4 by flow cytometry. Background technique [0002] Acute myeloid leukemia (AML) is a clonal disease of the hematopoietic system formed by the malignant transformation of pluripotent stem cells or hematopoietic progenitor cells at different stages of myeloid cell differentiation and development. AML is the most common type of acute leukemia, accounting for 2 / 3 of adult leukemia, and it shows an increasing trend with the aging of the population. AML is a highly heterogeneous disease. Abnormal blasts and immature cells (leukemic cells) in the bone marrow proliferate in large numbers and inhibit normal hematopoiesis at the time of onset, extensively infiltrating liver, spleen, lymph nodes and other organs, leading to anemia, Corresponding clinical manifestation...

Claims

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Application Information

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IPC IPC(8): G01N15/14G01N33/68G01N33/533
CPCG01N15/14G01N33/533G01N33/68
Inventor 吴庆宇严茹红董宁征
Owner SUZHOU UNIV
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