CN107807234A - A kind of method for detecting intracerebral microglia phagocytic activity - Google Patents
A kind of method for detecting intracerebral microglia phagocytic activity Download PDFInfo
- Publication number
- CN107807234A CN107807234A CN201711030933.5A CN201711030933A CN107807234A CN 107807234 A CN107807234 A CN 107807234A CN 201711030933 A CN201711030933 A CN 201711030933A CN 107807234 A CN107807234 A CN 107807234A
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- Prior art keywords
- phagocytic activity
- intracerebral
- emulsion particle
- microglia
- phagocyte
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
A kind of method for detecting intracerebral microglia phagocytic activity, it is related to the detection method of intracerebral microglia phagocytic activity.Diluted after intracerebral microglia is blown and beaten uniformly with complete medium, add the emulsion particle with fluorescence, after 4 hours are incubated, material in each orifice plate is cleaned, obtain supernatant and precipitation respectively；Precipitation is taken, the paraformaldehyde that mass percent is 4% is added and fixes cell, then nuclear staining is carried out with DAPI, is taken pictures under fluorescence microscope；Automatic Fluorescence Intensity Assays are carried out to photo again.Fluorescence intensity is higher, then phagocytic activity is stronger.Or supernatant is tested into absorbance with ELIASA.Absorbance is smaller, then the emulsion particle not swallowed is smaller, and phagocytic activity is stronger.The inventive method can be used for the power of the immunocompetence of accurate evaluation animal or human organism's phagocyte, while provide referential theoretical foundation to detect the cell biology detection method related to phagocyte.
Description
Technical field
The present invention relates to the association area of biocytology experiment, more particularly to macrophage and the small colloid of intracerebral in blood
The detection method of cell phagocytic activity.
Background technology
Detection on phagocyte phagocytic activity at present, both at home and abroad research concentrate on the inspection to phagocyte in animal body
Survey, monokaryon-phagocyte in the mostly circulatory system of detection（For not attached cell）, its detection process is as follows：
By the cellules such as chicken red blood cells injection animal abdominal cavity, animal is dissected afterwards and collects abdominal cavity phagocytic, passes through dyeing, mirror
Inspection, phagocytic activity of the observation phagocyte to chicken red blood cells.Gulped down finally by calculating percentage phagocytosis and phagocytic index measure
The phagocytic activity of phagocyte（Phagocytic rate=[cell number/neutrophil leucocyte of phagocytosis bacterium is total (200)] × 100%；Phagocytosis
Index=(200 grain bacteriums gulp down bacterium sum/200 neutrophil leucocytes) × 100%）.
The shortcomings that above method is：1. being only limitted to animal vivo detection, by animal individual differentia influence, can not standardize.
2. being influenceed by other intraperitoneal heteroproteose cells, the phagocyte of high-purity can not be obtained, observation index is difficult.3. it can not be directed to single
Only a certain phagocyte is detected.4. the cellule swallowed in Microscopic observation, causes leakage to be counted because iuntercellular is overlapping
Number, influence to calculate precision.5. operated with animal, Medicine Ethics and experimenter's safety.
The content of the invention
The invention aims to overcome the deficiencies in the prior art, there is provided one kind is simple to operate, can objectively respond cell gulps down
The method for biting the detection intracerebral microglia phagocytic activity of practical capacity
The present invention comprises the following steps：
1）Intracerebral microglia is fully blown and beaten uniformly with containing the complete medium that serum is 10%, then diluted with culture medium
After plant in orifice plate；After 24 hours, then the emulsion particle with fluorescence is added in the orifice plate and is incubated；
2）Incubation process was cleaned after 4 hours to material in each orifice plate, obtained supernatant and precipitation respectively；
3）Precipitation is taken, the paraformaldehyde that mass percent is 4% is added and fixes cell, then nuclear staining is carried out with DAPI, then glimmering
Taken pictures under light microscope；Automatic Fluorescence Intensity Assays are carried out to photo again.
Fluorescence intensity is higher, illustrates that phagocytic activity is stronger.
Or supernatant is tested into absorbance with ELIASA.Absorbance is smaller, then the emulsion particle not swallowed is smaller,
It is stronger that phagocytic activity is reflected indirectly.
Through repetition test, the above is incubated process by 4 hours peaks that can reach the phagocytosis of intracerebral microglia, is reaching
When peak swallows effect, cell is fixed using paraformaldehyde, the fluorescent latex particles that can prevent from swallowing are lost in.
The related detecting method that there is no isolated cells phagocytic activity to detect at present, advantages of the present invention are embodied in lower section
Method：
1. being the detection to " isolated cells ", cell is single, it is easier to standardizes.
2. the phagocytic activity of such as microglia " attached cell " can be detected.
3. using a diameter of 2 microns of fluorescent latex particles, phagocyte is swallowed fluorescent latex particles, be easy to observation and
Calculate.
4. using the method for calculating fluorescence intensity and supernatant absorbance, avoid in original method by Phagocytic granules vision
It is overlapping to cause counting loss, reach criterion calculation.
5. avoiding injecting chicken red blood cells etc. in animal body, using direct in vitro culture, streamline operation, avoid noting
Penetrating animal causes operator injured, avoids the pain of animal, improves efficiency, shortens the time.
6. detection method more than, the adherent phagocyte that can be expanded in other tissues and the phagocytosis of the circulatory system are thin
Born of the same parents.The inventive method can be used for the power for assessing the immunocompetence of animal or human organism's phagocyte, while be detection and phagocytosis
The related cell biology detection method of cell provides referential theoretical foundation.
Further, a diameter of 2 μm of emulsion particle of the present invention.Through repeatedly checking is found repeatedly, if emulsion particle is straight
Footpath is excessive, and the phagocytosis of phagocyte can be caused difficult；, whereas if emulsion particle diameter is too small, then can cause to observe and count
It is difficult.
Embodiment
1st, using known public succusion or trypsin digestion, the microglia of acquisition SD rats.
2nd, microglia is fully blown and beaten uniformly with containing the complete medium that serum is 10%, then diluted with culture medium
It is 2 × 10 to cell concentration5/ ml, plant respectively in 24 orifice plates, per hole 0.5ml.
3rd, after 24 hours, a diameter of 2 μm of emulsion particles of the 5 μ L with red fluorescence is separately added into each hole, and continue
It is incubated 4 hours.Tests prove that：Reach within 4 hours phagocytosis peak.
4th, after phagocytosis in 4 hours, cleaning, supernatant and precipitation are obtained respectively.
5th, precipitation is taken, fixes cell with the paraformaldehyde that mass fraction is 4%, the fluorescent latex particles for preventing from swallowing are lost in.
Nuclear staining is carried out with DAPI again, then whether plays phagocytic function, and the latex of phagocytosis in fluorescence microscopy Microscopic observation cell
The number of particle number is simultaneously taken pictures.
The calculating of cell phagocytic activity
Average fluorescent strength is calculated using individual photo analysis under fluorescence microscope：Each photo is carried out with softwares such as image J
Automatic Fluorescence Intensity Assays, the emulsion particle red fluorescence intensity of each photo is obtained respectively.
And the emulsion particle red fluorescence intensity of the last 24 photo is calculated, draw average fluorescent strength.
As average fluorescent strength is higher, then illustrate that phagocytic activity is higher.
6th, in addition, can also be tested respectively with ELIASA 24 portions of supernatants, point separately 24 parts of absorbances of acquirement, then calculate flat
Equal absorbance.
As absorbance is lower, then the emulsion particle not swallowed is fewer, and this also illustrates that microglia gulps down indirectly
It is stronger to bite ability.
Claims (2)
- A kind of 1. method for detecting intracerebral microglia phagocytic activity, it is characterised in that comprise the following steps：1）Intracerebral microglia is fully blown and beaten uniformly with containing the complete medium that serum is 10%, then diluted with culture medium After plant in orifice plate；After 24 hours, then the emulsion particle with fluorescence is added in the orifice plate and is incubated；2）Incubation process was cleaned after 4 hours to material in each orifice plate, obtained supernatant and precipitation respectively；3）Precipitation is taken, the paraformaldehyde that mass percent is 4% is added and fixes cell, then nuclear staining is carried out with DAPI, then glimmering Taken pictures under light microscope；Automatic Fluorescence Intensity Assays are carried out to photo again；Or supernatant is tested into absorbance with ELIASA.
- 2. according to the method for claim 1, it is characterised in that：A diameter of 2 μm of the emulsion particle.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711030933.5A CN107807234A (en) | 2017-10-30 | 2017-10-30 | A kind of method for detecting intracerebral microglia phagocytic activity |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711030933.5A CN107807234A (en) | 2017-10-30 | 2017-10-30 | A kind of method for detecting intracerebral microglia phagocytic activity |
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| CN107807234A true CN107807234A (en) | 2018-03-16 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108872562A (en) * | 2018-09-19 | 2018-11-23 | 甄勇 | A kind of kit detecting adherent phagocyte phagocytic activity |
| RU214554U1 (en) * | 2022-05-25 | 2022-11-03 | Федеральное государственное бюджетное образовательное учреждение высшего образования Санкт-Петербургский государственный университет ветеринарной медицины ФГБОУ ВО СПбГУВМ | Device for determining the phagocytic activity of alveolar macrophages |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0134707A2 (en) * | 1983-08-09 | 1985-03-20 | Toray Industries, Inc. | Method of assaying the activity of cells |
| JPS60131462A (en) * | 1983-12-21 | 1985-07-13 | Toray Ind Inc | Fine particle for measuring cell function |
| US4581334A (en) * | 1983-04-25 | 1986-04-08 | Ortho Diagnostics Systems, Inc. | Simultaneous detection of leukocyte phagocytic and killing ability |
| JPS61128167A (en) * | 1984-11-26 | 1986-06-16 | Hitachi Chem Co Ltd | Determination of predation capacity of biophagous cell |
| AT396941B (en) * | 1990-01-08 | 1993-12-27 | Kohlweg Ernst Dr | Method for the determination of cells with phagocytotic activity |
| WO2005062043A1 (en) * | 2003-12-22 | 2005-07-07 | Universität Ulm | Examination method for phagocytosis |
| CN101975853A (en) * | 2010-11-26 | 2011-02-16 | 天津农学院 | Method for detecting phagocytic rate of blood cells by fluorescence method |
| CN103710306A (en) * | 2012-10-09 | 2014-04-09 | 复旦大学附属华山医院 | Method used separating and purifying microglia cells |
| CN103994963A (en) * | 2014-04-29 | 2014-08-20 | 中国水产科学研究院东海水产研究所 | Method used for detecting ostrea plicatula eosinophilic and basophilic granulocyte phagocytic ability |
| CN106950203A (en) * | 2015-10-15 | 2017-07-14 | 浜松光子学株式会社 | Phagocytic activity evaluation method and fluorescence analysis |
| CN107192656A (en) * | 2017-04-06 | 2017-09-22 | 大连工业大学 | A kind of coelomocyte of Apostichopus japonicus phagocytic rate detection method based on flow cytometer |
| CN107208062A (en) * | 2015-01-16 | 2017-09-26 | 新加坡科技研究局 | From pluripotent stem cell differentiation macrophage |
-
2017
- 2017-10-30 CN CN201711030933.5A patent/CN107807234A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4581334A (en) * | 1983-04-25 | 1986-04-08 | Ortho Diagnostics Systems, Inc. | Simultaneous detection of leukocyte phagocytic and killing ability |
| EP0134707A2 (en) * | 1983-08-09 | 1985-03-20 | Toray Industries, Inc. | Method of assaying the activity of cells |
| JPS60131462A (en) * | 1983-12-21 | 1985-07-13 | Toray Ind Inc | Fine particle for measuring cell function |
| JPS61128167A (en) * | 1984-11-26 | 1986-06-16 | Hitachi Chem Co Ltd | Determination of predation capacity of biophagous cell |
| AT396941B (en) * | 1990-01-08 | 1993-12-27 | Kohlweg Ernst Dr | Method for the determination of cells with phagocytotic activity |
| WO2005062043A1 (en) * | 2003-12-22 | 2005-07-07 | Universität Ulm | Examination method for phagocytosis |
| CN101975853A (en) * | 2010-11-26 | 2011-02-16 | 天津农学院 | Method for detecting phagocytic rate of blood cells by fluorescence method |
| CN103710306A (en) * | 2012-10-09 | 2014-04-09 | 复旦大学附属华山医院 | Method used separating and purifying microglia cells |
| CN103994963A (en) * | 2014-04-29 | 2014-08-20 | 中国水产科学研究院东海水产研究所 | Method used for detecting ostrea plicatula eosinophilic and basophilic granulocyte phagocytic ability |
| CN107208062A (en) * | 2015-01-16 | 2017-09-26 | 新加坡科技研究局 | From pluripotent stem cell differentiation macrophage |
| CN106950203A (en) * | 2015-10-15 | 2017-07-14 | 浜松光子学株式会社 | Phagocytic activity evaluation method and fluorescence analysis |
| CN107192656A (en) * | 2017-04-06 | 2017-09-22 | 大连工业大学 | A kind of coelomocyte of Apostichopus japonicus phagocytic rate detection method based on flow cytometer |
Non-Patent Citations (1)
| Title |
|---|
| 闫可: "骨髓间充质干细胞调节小胶质细胞功能作用的实验研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108872562A (en) * | 2018-09-19 | 2018-11-23 | 甄勇 | A kind of kit detecting adherent phagocyte phagocytic activity |
| RU214554U1 (en) * | 2022-05-25 | 2022-11-03 | Федеральное государственное бюджетное образовательное учреждение высшего образования Санкт-Петербургский государственный университет ветеринарной медицины ФГБОУ ВО СПбГУВМ | Device for determining the phagocytic activity of alveolar macrophages |
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Application publication date: 20180316 |
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