CN113293192A

CN113293192A - Neutrophil chemotaxis detection kit

Info

Publication number: CN113293192A
Application number: CN202110599037.0A
Authority: CN
Inventors: 孙炳伟
Original assignee: Suzhou Municipal Hospital North District
Current assignee: Suzhou Municipal Hospital North District
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2021-08-24

Abstract

The application relates to a neutrophil chemotaxis detection kit, which comprises a reagent R1 and a reagent R2 which are independent of each other, wherein the reagent R1 consists of the following substances: RIPM 1640 medium 5-10 ml; 3-7ml of sterilized water for injection; 10 Xbuffer solution containing Ca2+, Mg2+ HBSS 1-3 ml; 3-7ml of fetal bovine serum FBS; the reagent R2 is composed of the following substances: 10-20ml of PolymorphPrep neutrophilic granulocyte separating medium; 10-20ml of a neutrophil culture medium; chemotaxis fMLP10-30 ul. The extracted neutrophils can be directly placed in the reagent kit, so that the complexity of the detection procedure is avoided, and the detection result is quickly obtained.

Description

Neutrophil chemotaxis detection kit

[ technical field ] A method for producing a semiconductor device

The application relates to a neutrophil chemotaxis detection kit, belonging to the technical field of biological detection.

[ background of the invention ]

Neutrophils are important innate immune cells for resisting pathogen invasion of a human body, are produced by bone marrow hematopoietic stem cells, are differentiated and developed in bone marrow and enter blood or tissues, are white blood cells with the largest quantity in peripheral blood of an organism, and account for 40% -75% of the total number of the white blood cells under a normal physiological state. In the ruin staining smear, the cytoplasm of neutrophils is colorless or light red, the nucleus of neutrophils is rod-shaped or 2-5 leaf-shaped, and filaments are connected between leaves, so that the neutrophils are also called polymorphonuclear leukocytes (PMNs) and have chemotactic, phagocytic and bactericidal functions. When a human body is attacked by pathogenic bacteria, the neutrophil can chemotaxis to an infected part, engulf the bacteria into the cell and kill the bacteria through multiple ways, is the first line of defense of the human body against bacterial infection and plays an important role in a non-specific immune system of the human body.

In recent years, a large number of domestic and foreign studies show that various diseases such as infection, severe disease, tumor, diabetes and the like are closely related to neutrophils: the chemotaxis of the neutrophils of the patient is abnormal, so that the neutrophils can not reach the infection part to remove pathogenic microorganisms, and meanwhile, excessive neutrophils are recruited to the non-inflammation part to cause organ damage. Therefore, the detection and analysis of neutrophil chemotaxis provides a new direction for the accurate diagnosis and treatment of immune conditions of patients, and has very important clinical significance.

Accordingly, there is a need for improvements in the art that overcome the deficiencies in the prior art.

[ summary of the invention ]

The present application aims to provide a neutrophil chemotaxis assay kit which can solve the above-mentioned problems of the prior art and can rapidly detect the chemotactic function of neutrophils.

The purpose of the application is realized by the following technical scheme: the neutrophil chemotaxis detection kit comprises a reagent R1 and a reagent R2 which are independent of each other, wherein the reagent R1 consists of the following substances: RIPM 1640 medium 5-10 ml; 3-7ml of sterilized water for injection; 10 Xbuffer solution containing Ca2+, Mg2+ HBSS 1-3 ml; 3-7ml of fetal bovine serum FBS;

the reagent R2 is composed of the following substances: 10-20ml of PolymorphPrep neutrophilic granulocyte separating medium; 10-20ml of a neutrophil culture medium; chemotaxis fMLP10-30 ul.

In one embodiment, the neutrophil medium in the reagent R2 comprises 1640 medium of 10% FBS.

In one embodiment, the fetal bovine serum FBS and the chemoattractant fMLP are stored at a temperature of-20 ℃.

In one embodiment, the neutrophil chemotaxis assay kit further comprises a cell culture dish and agarose powder which can be formed in the cell culture dish by a dissolving mode, wherein the weight of the agarose powder is 0.1-0.3 g;

wherein the reagent R1, the cell culture dish and agarose powder form a first kit.

In one embodiment, the number of the cell culture dishes is at least 5.

In one embodiment, the neutrophil chemotaxis assay kit further comprises sterile gauze;

wherein the reagent R2 forms a second kit with the sterile gauze.

In one embodiment, the neutrophil chemotaxis assay kit further comprises an agarose gel punch which is provided independently from the first kit and the second kit.

Compared with the prior art, the method has the following beneficial effects: the extracted neutrophils can be directly placed in the reagent kit, so that the complexity of the detection procedure is avoided, and the detection result is quickly obtained.

[ description of the drawings ]

FIG. 1 is a schematic representation of neutrophil chemotaxis in a healthy human using the neutrophil chemotaxis assay kit of the present application.

FIG. 2 is a schematic representation of neutrophil chemotaxis in ICU critically ill patients using the neutrophil chemotaxis assay kit of the present application.

FIG. 3 is a schematic representation of neutrophil chemotaxis of tumor patients using the neutrophil chemotaxis assay kit of the present application.

[ detailed description ] embodiments

In order to make the aforementioned objects, features and advantages of the present application more comprehensible, embodiments accompanying the present application are described in detail below with reference to the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the application and are not limiting of the application. It should be further noted that, for the convenience of description, only some of the structures related to the present application are shown in the drawings, not all of the structures. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The terms "comprising" and "having," as well as any variations thereof, in this application are intended to cover non-exclusive inclusions. For example, a process, method, system, article, or apparatus that comprises a list of steps or elements is not limited to only those steps or elements listed, but may alternatively include other steps or elements not listed, or inherent to such process, method, article, or apparatus.

Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. It is explicitly and implicitly understood by one skilled in the art that the embodiments described herein can be combined with other embodiments.

Example 1

The neutrophil chemotaxis detection kit comprises a reagent R1 and a reagent R2 which are independent of each other, wherein the reagent R1 consists of the following substances: RIPM 1640 medium 9.2 ml; 4.6ml of sterilized water for injection; 10 × 1.15ml of buffer solution containing Ca2+, Mg2+ HBSS; 4.6ml of fetal bovine serum FBS; reagent R2 consisted of: 15ml of PolymorphPrep neutrophile separation solution; 15ml of neutrophil medium; chemoattractant fMLP20 ul. Wherein the concentration of the component in reagent R2 is the final concentration of the component in the reagent, and the neutrophil medium in reagent R2 comprises 1640 medium of 10% FBS.

Wherein the preservation temperature of the fetal bovine serum FBS and the chemotactic compound fMLP is-20 ℃.

The neutrophil chemotaxis detection kit also comprises a cell culture dish and agarose powder which can be formed in the cell culture dish in a dissolving mode, wherein the weight of the agarose powder is 0.275 g; thus, reagent R1 and cell culture dish, agarose powder form the first kit. The number of the cell culture dishes is at least 5.

The neutrophil chemotaxis detection kit also comprises sterile gauze, the number of the sterile gauze is consistent with that of the cell culture dish, and the number of the sterile gauze is at least 5; thus, reagent R2 forms a second kit with sterile gauze.

The neutrophil chemotaxis detection kit also comprises an agarose gel puncher, and the agarose gel puncher is independently arranged with the first kit and the second kit.

The neutrophil is a suspension cell, and the kit provided by the application is used for detecting the chemotactic function of the neutrophil, so that the neutrophil can be attached to the bottom plane of a cell culture dish to perform chemotactic movement and can be visually observed under a microscope, and the kit is convenient and fast.

Please refer to fig. 1 to 3, which are the neutrophil chemotactic functions detected by the kit of the present application in healthy people, ICU critically infected patients and tumor patients, respectively.

In fig. 1, it can be seen that the chemotactic function of neutrophils in healthy people is complete, the reaction to the chemokine fMLP is rapid, the chemotactic movement can be performed efficiently, and after 2h of chemotactic movement, the chemotactic distance is far, the number of cells performing chemotactic movement is large, and the chemotactic index is high.

As can be seen in FIG. 2, the chemotactic function of neutrophils in ICU critically ill infected patients is obviously impaired, the chemotactic effect is insensitive to the response of the chemokine fMLP, after 2h chemotactic movement, the chemotactic distance is shorter, the number of cells undergoing chemotactic movement is obviously reduced, and the chemotactic index is lower.

As can be seen in FIG. 3, the chemotactic function of the neutrophils of the tumor patients is also impaired, the chemotactic function is insensitive to the response of the chemokine fMLP, and after 2h of chemotactic movement, the chemotactic distance is short, the number of cells undergoing chemotactic movement is obviously reduced, and the chemotactic index is obviously reduced.

The Chemotactic Distance (CD) mentioned above is: neutrophils chemotactic for the greatest distance that can be reached in the agarose chemotaxis model for two hours. Unit: micron (um).

Percentage of chemotactic cells (Chemo Cell Ratio, CCR) is: the total number of chemotactic cells was a percentage of the total number of chemotactic cells (105).

The chemotaxis Index (Chemo Index, CI) is: the number of chemotactic cells in the region I and the region II is the ratio of the number of the total chemotactic cells.

In summary, the following steps:

the above is only one specific embodiment of the present application, and any other modifications based on the concept of the present application are considered as the protection scope of the present application.

Claims

1. The neutrophil chemotaxis detection kit is characterized by comprising a reagent R1 and a reagent R2 which are independent of each other, wherein the reagent R1 consists of the following substances: RIPM 1640 medium 5-10 ml; 3-7ml of sterilized water for injection; 10 Xbuffer solution containing Ca2+, Mg2+ HBSS 1-3 ml; 3-7ml of fetal bovine serum FBS;

2. The neutrophil chemotaxis assay kit according to claim 1, wherein the neutrophil culture medium in the reagent R2 comprises 1640 medium of 10% FBS.

3. The neutrophil chemotaxis assay kit according to claim 1, wherein the preservation temperature of said fetal bovine serum FBS and said chemotactic agent fMLP is-20 ℃.

4. The neutrophil chemotaxis assay kit according to claim 1, further comprising a cell culture dish and agarose powder which can be shaped in the cell culture dish by means of dissolution, wherein the agarose powder has a weight of 0.1-0.3 g;

5. The neutrophil chemotaxis assay kit according to claim 4, wherein the number of the cell culture dishes is at least 5.

6. The neutrophil chemotaxis assay kit of claim 4, further comprising sterile gauze;

wherein the reagent R2 forms a second kit with the sterile gauze.

7. The neutrophil chemotaxis assay kit according to claim 6, further comprising an agarose gel punch provided separately from the first and second kits.