CN104293644A - Assisting part for blood monocyte separation - Google Patents
Assisting part for blood monocyte separation Download PDFInfo
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- CN104293644A CN104293644A CN201410300855.6A CN201410300855A CN104293644A CN 104293644 A CN104293644 A CN 104293644A CN 201410300855 A CN201410300855 A CN 201410300855A CN 104293644 A CN104293644 A CN 104293644A
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- lifting rope
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/08—Flask, bottle or test tube
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M45/00—Means for pre-treatment of biological substances
- C12M45/05—Means for pre-treatment of biological substances by centrifugation
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Abstract
The invention discloses an assisting part for blood monocyte separation. The assisting part has a semi-isolation effect, is used to restrict the adding speed of a liquid, and is a buffer block for preventing the too fast dropping speed of the liquid. The buffer block is composed of a cylindrical buffer pedestal, a conical operation structure and an aseptic lifting rope hole, wherein the cylindrical buffer pedestal and the conical operation structure form one body, the diameter of the cylindrical buffer pedestal is 1-3mm smaller than the diameter of the inner wall of a centrifuge tube to guarantee that blood flows between the cylindrical buffer pedestal and the centrifuge to a low position, an angle between the bus and the bottom surface of the conical operation structure is 55-65DEG, and the aseptic lifting rope hole is provided with an aseptic lifting rope for moving the buffer block. The assisting part has a buffer effect, and blood must flows through a slit between the buffer block and the wall of the centrifuge tube to the surface of a lymphocyte separation liquid, so the influence of the too fast dropping speed of blood on liquid layering is prevented.
Description
Technical field
The present invention relates to cellular segregation field, the accessory of specifically a kind of blood mononuclear cell separation.
Background technology
Monocyte (English: Monocyte) is a kind of white corpuscle in human immune system.The hemopoietic stem cell of cells of monocytic origin in marrow, and grow in marrow.Still immature thin monocyte born of the same parents are remained when they enter blood from marrow.Compare with other hemocytes, containing more non-specific lipase in monocyte, and there is stronger phagolysis.Monocyte moves in surrounding tissue after stopping 2-3 days in blood, and cell volume continues to increase, and diameter can reach 50-80 μm, and lysosome particle contained in cell and mitochondrial number also increase, and becomes ripe cell.Fixing monocyte is in the tissue called tissue macrophages, and they are often present in the organs such as lymphoglandula, alveolus wall, marrow, liver and spleen in a large number.The monocyte that have activated and tissue macrophages can generate and discharge various kinds of cell poison, Interferon, rabbit and interleukin-, participate in body defense mechanism, also produce the factor that some can promote endotheliocyte and smooth muscle cell growth.Around inflammation, monocyte can carry out cell fission, and surrounds foreign matter.Monocyte (monocytes) is the maximum white corpuscle of volume.The normal off normal of its nucleus, in polymorphism, as oval, kidney shape, the shape of a hoof, irregular shape etc., often has folding sense; Chromatin is in loose netted, painted more shallow.Kytoplasm is more, basophilia, but because dying dusty blue containing azurophilic granule tiny in a large number, particle is containing peroxidase.
In medical science and biologically, monocyte is the source of immunocyte and blood stem cell, and for this reason, isolation technique is rapidly developed, and existing the most frequently used monocyte isolation technique is exactly density gradient zonal centrifugation method (abbreviation zonal centrifugation).
Density gradient centrifugation is added in inertia gradient media by sample to carry out centrifugal settling or sedimentation equilibrium, under certain centrifugal force, particle to be assigned in gradient on some specific position, to form the separation method of different zone.The advantage of this method is: 1. good separating effect, can once obtain purer particle; 2. wide accommodation, can be separated the particle with settling ratio difference, can be separated again the particle of certain buoyant density difference as differential centrifugation; 3. particle can not crimp, can keep seed activity, and prevent established zone from causing mixing due to convection current.The shortcoming of this method is: 1. centrifugation time is longer; 2. preparation inertia gradient media solution is needed; 3. operation is strict, not easily grasps.
Density gradient centrifugation is also known as density gradient equilibrium centrifugation.With ultracentrifuge to small-molecule substance solution, add a centrifuge field for a long time and reach sedimentation equilibrium, in settling bowl, from liquid level to bottom, occur certain density gradient.If add a small amount of macromolecular solution in this solution, then the part that solution internal ratio solvent density is large just produces macromole sedimentation, and the part less than solvent density will float, and finally in the position of gravity and buoyant equilibrium, gathers and forms macromole ribbon.Utilize this phenomenon, measure the density of swimming of nucleic acid or protein etc., or carry out a kind of sedimentation equilibrium method of analyzing according to its difference.From meter Xi Erxun (M.Meselson) in 1958, Si Taer (F.W.Stahl), since Wei Nuoge rad (J.Vinograd) has successfully been separated (15N) DNA and (14N) DNA, this method has obtained many achievements.For obtaining necessary concentration gradient, the dense cesium chloride solution of many employings, so sometimes also use this title of Cesium chloride concentrations gradient centrifugation, also can adopt the solution such as rubidium chloride, cesium bromide.Usually utilize analytical ultracentrifuge, but cell granulations composition being carried out be separated situation about waiting for the purpose of purifying, utilizing density difference, using and be separated ultracentrifuge, adopt the density gradient of previously prepared good sucrose etc.(2) adopt some small molecule solution such as sucrose, in the sample ground being separated ultracentrifuge, prepare density gradient in advance, after adding a small amount of macromolecular solution of one deck in the above, centrifugal, macromole sedimentation with regard to formation stratiform.If containing many compositions that settling ratio is different, just there will be many layers.This situation adopts suitable layout number, takes out the solution in sample pool, then studies.This is a kind of sedimentation velocity method different from (1), except being used to except common sedimentation velocity method with identical object, has superiority can take out on this aspect of isolate.Because adopting sucrose density gradient, so be also called sucrose density gradient centrifugation more.
Principle: when there is settling ratio difference between variable grain, under certain centrifugal action, particle, separately with certain speed sedimentation, density gradient different zones forms the method for zone.Dielectric gradient should be pre-formed, and the maximum density of medium is less than the density of all samples particle.Conventional has sucrose, glycerine; The preparation gradient mixer of density gradient liquid, is formed by the density gradient progressively raised at the bottom of the mouth of pipe to pipe.
Step
1. in centrifuge tube, add appropriate lymphocyte separation medium.
2. taking heparin anti-freezing venous blood and normal saline fully mix, and are slowly superimposed on laminated fluid level along tube wall with dropper, note keeping clearly interface.Horizontal centrifugal 2000rpm × 20 minute.
3. be divided into three layers in centrifugal rear pipe, upper strata is the mixed solution of blood plasma and physiological saline, and lower floor is mainly red corpuscle and granulocyte.Middle level is lymphocyte separation medium, and have a white cloud and mist layer narrow band based on mononuclearcell in upper, interface, middle level, this one deck is exactly mononuclearcell, comprises lymphocyte and monocyte.In addition, also containing thrombocyte.
4. be inserted into cloud and mist layer with capillary vessel, draw mononuclearcell.Insert in another brachymedial pipe, add 5 times with the physiological saline of upper volume, 1500rpm × 10 minute, washed cell twice.
5. after final centrifugation, abandon supernatant, add the RPMI1640 containing 10% foetal calf serum, re-suspended cell.Get a cell suspension and expect that blue dye liquor mixes with one 0.2%, on blood counting chamber, count the total cellular score in four block plaid.
Also there are problems in existing density gradient centrifugation: 1, operation steps cannot be streamlined any further, and cannot carry out intelligent automation operation; 2, centrifugation time is oversize; 3, when liquid feeding, lymphocyte separation medium is added in bottom centrifuge tube, and blood is added in upper strata, but due to operation reason, often occurs that liquid feeding speed is too fast, and a large amount of blood and lymphocyte separation medium mixing, affect final separating effect.
In order to solve above problem, the Leucosep of greiner Brand Design
tMseparator tube, its core technology is in the polypropylene centrifuge tube of high-clarity, add a porous sieve plate.This sieve plate is made up of the polyethylene of high standard, carrys out isolated cell by the pore size strictly controlling sieve plate, substantially reduces the time of sample process.Directly the anticoagulation collected or sample of bone marrow can be poured in LeucosepTM separator tube.Porous sieve plate can prevent the mixing in advance of sample and parting liquid.Due to the difference of various blood cell density, in centrifugal process, lymphocyte and monocyte all can separate with red corpuscle and granulocyte, and lymphocyte and monocyte can in the upper strata enrichments of parting liquid.
Summary of the invention
Density gradient centrifugation is existing main blood mononuclear cell isolation technique, the difficulty that possesses skills is low, advantage simple to operate, but first lymphocyte separation medium must be added bottom, slowly blood is added in above lymphocyte separation medium again, obvious layering can be formed, thus the separating effect reached after centrifugal treating, but often due to firmly excessive during operation, blood and lymphocyte separation medium is caused to mix, affect final separating effect, for this reason, the invention provides the accessory that a kind of blood mononuclear cell is separated, be covered in lymphocyte separation medium surface, thus anti-Hemostatic Oral Liquid to add hourly velocity too fast, reach the object preventing two kinds of liquid mixing.
For achieving the above object, the invention provides following technical scheme:
The accessory that a kind of blood mononuclear cell is separated, be one and play half buffer action, for carrying out speed limit to liquid feeding, prevent from dripping the too fast buffer block of liquid hourly velocity, this buffer block is by cylindrical buffer base, conical operating structure and aseptic lifting rope hole composition, wherein cylindrical buffer base, conical operating structure is as a whole, the diameter 1-3mm less of centrifuge tube inner diameter of described cylindrical buffer base, ensure that blood can from below flowing between cylindrical buffer base and centrifuge tube, bus and the bottom surface angle of conical operating structure are 55-65 °, it aseptic lifting rope hole is an aseptic lifting rope for mobile cushioning block, the material of described buffer block and aseptic lifting rope, be any there is hydrophobicity, material that no cytotoxicity, density are less than lymphocyte separation medium, water and blood.
1., the diameter 2mm less of centrifuge tube inner diameter of described cylindrical buffer base 3 as the further scheme of the present invention: described buffer block has following characteristics:, blood can from below centre flows to; 2., density is less than water, ensures that density is less than lymphocyte separation medium and blood, thus can float on blood sample after ensureing application of sample; 3., be hydrophobic material, lymphocyte separation medium, blood and water can not be stained with; 4., smooth surface; 5., nontoxic to biomass cells.
As the further scheme of the present invention: the bus of conical operating structure and bottom surface angle are 60 °.
1., smooth as the further scheme of the present invention: the smooth diameter of described aseptic lifting rope is 1mm cord, and has following characteristics:; 2., hydrophobic; 3., to cytotoxic; 4., density is consistent with buffer block material, or is less than the density of Buffer Unit material; Thus can float on blood sample after guarantee application of sample; Described aseptic lifting rope by buffer block shift-in centrifuge tube, and can shift out buffer block after application of sample completes from centrifuge tube.
As the further scheme of the present invention: described centrifuge tube is 15ml or 50ml Standard centrifugal pipe.
Existing density gradient centrifugation mainly uses 15ml and the 50ml centrifuge tube in laboratory to carry out, in order to save operation, the operation skill of people is relied on to carry out liquid feeding completely, this can cause in face of great many of experiments, technician is in order to the time of pursuing, improve the layering that operating speed have ignored lymphocyte separation medium and blood, compared with prior art, the invention has the beneficial effects as follows: accessory of the present invention can play shock absorption, blood must flow to the surface of lymphocyte separation medium by the gap of buffer block and centrifugal tube wall, prevent the too fast impact on liquid layered of blood sample rate of addition.The present invention also has the following advantages:
1, cost is low, the most design abroad of existing special centrifuge tube, expensive, be not suitable for extensive medical treatment or experiment use, and this product is cheap;
2, processing requirement is lower, and the product of this design is processed without the need to high precision, makes structure simple;
3, use handiness strong, any common centrifuge tube, comprises 15ml and 50ml centrifuge tube, as long as the body of standard, can use corresponding assembly, simultaneously for the centrifuge tube of special diameter, only need diameter to modify just can produce for product.
Accompanying drawing explanation
Fig. 1 is the front view of the accessory that blood mononuclear cell is separated;
Fig. 2 is the left view of the accessory that blood mononuclear cell is separated;
Fig. 3 is the vertical view of the accessory that blood mononuclear cell is separated;
Fig. 4 is the using state figure of the accessory that blood mononuclear cell is separated.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Refer to Fig. 1 ~ 4, in the embodiment of the present invention, the accessory that a kind of blood mononuclear cell is separated, be one and play half buffer action, for carrying out speed limit to liquid feeding, prevent from dripping the too fast buffer block of liquid hourly velocity, this buffer block is by cylindrical buffer base 3, conical operating structure 2 and aseptic lifting rope hole 1 form, wherein cylindrical buffer base 3, conical operating structure 2 is as a whole, the diameter 1-3mm less of centrifuge tube 5 inner diameter of described cylindrical buffer base 3, ensure that blood can from below flowing between cylindrical buffer base 3 and centrifuge tube 5, bus and the bottom surface angle of conical operating structure 2 are 55-65 °, the best is 60 °, it aseptic lifting rope hole 1 is an aseptic lifting rope 4 for mobile cushioning block, the material of described buffer block and aseptic lifting rope 4, be any there is hydrophobicity, material that no cytotoxicity, density are less than lymphocyte separation medium, water and blood.
1., the diameter 2mm less of centrifuge tube 5 inner diameter of described cylindrical buffer base 3 described buffer block has following characteristics:, and blood can from below centre flows to; 2., density is less than water, ensures that density is less than lymphocyte separation medium and blood, thus can float on blood sample after ensureing application of sample; 3., be hydrophobic material, lymphocyte separation medium, blood and water can not be stained with; 4., smooth surface; 5., nontoxic to biomass cells.1., smooth the smooth diameter of described aseptic lifting rope 4 is 1mm cord, and has following characteristics:; 2., hydrophobic; 3., to cytotoxic; 4., density is consistent with buffer block material, or is less than the density of Buffer Unit material; Thus can float on blood sample after guarantee application of sample; Described aseptic lifting rope 4 by buffer block shift-in centrifuge tube 5, and can shift out buffer block after application of sample completes from centrifuge tube 5.Described centrifuge tube 5 is 15ml or 50ml Standard centrifugal pipe.
The present invention places the hydrophobic buffer block of low density of a blocks design above lymphocyte separation medium, prevents dripping blood hourly velocity too fast, causes liquid mixing; Because buffer block density is lower, after adding blood, blood flows to above lymphocyte separation medium by the space between buffer block and centrifugal tube wall, and buffer block is floated, and final buffer block can take out, and can not impact centrifugation; Buffer block adopts hydrophobic material to make, and does not have above blood or lymphocyte separation medium be attached to, does not affect amount of liquid; Buffer block is no cytotoxicity material, can not produce any impact to cell, does not affect follow-up cultivation and uses; Low in input cost, simple to operate.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.Any Reference numeral in claim should be considered as the claim involved by limiting.
In addition, be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should by specification sheets integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
Claims (5)
1. the accessory of a blood mononuclear cell separation, it is characterized in that, be one and play half buffer action, for carrying out speed limit to liquid feeding, prevent from dripping the too fast buffer block of liquid hourly velocity, this buffer block is by cylindrical buffer base, conical operating structure and aseptic lifting rope hole composition, wherein cylindrical buffer base, conical operating structure is as a whole, the diameter 1-3mm less of centrifuge tube inner diameter of described cylindrical buffer base, ensure that blood can from below flowing between cylindrical buffer base and centrifuge tube, bus and the bottom surface angle of conical operating structure are 55-65 °, it aseptic lifting rope hole is an aseptic lifting rope for mobile cushioning block, the material of described buffer block and aseptic lifting rope, be any there is hydrophobicity, material that no cytotoxicity, density are less than lymphocyte separation medium, water and blood.
2. the accessory of blood mononuclear cell separation according to claim 1, it is characterized in that, 1., the diameter 2mm less of centrifuge tube inner diameter of described cylindrical buffer base 3 described buffer block has following characteristics:, and blood can from below centre flows to; 2., density is less than water, ensures that density is less than lymphocyte separation medium and blood, thus can float on blood sample after ensureing application of sample; 3., be hydrophobic material, lymphocyte separation medium, blood and water can not be stained with; 4., smooth surface; 5., nontoxic to biomass cells.
3. the accessory of blood mononuclear cell separation according to claim 1, it is characterized in that, the bus of conical operating structure and bottom surface angle are 60 °.
4. the accessory that is separated of blood mononuclear cell according to claim 1,1., smooth it is characterized in that, the smooth diameter of described aseptic lifting rope is 1mm cord, and has following characteristics:; 2., hydrophobic; 3., to cytotoxic; 4., density is consistent with buffer block material, or is less than the density of Buffer Unit material; Thus can float on blood sample after guarantee application of sample.
5. the accessory of blood mononuclear cell separation according to claim 1, it is characterized in that, described centrifuge tube is 15ml or 50ml Standard centrifugal pipe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410300855.6A CN104293644A (en) | 2014-06-24 | 2014-06-24 | Assisting part for blood monocyte separation |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410300855.6A CN104293644A (en) | 2014-06-24 | 2014-06-24 | Assisting part for blood monocyte separation |
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| Publication Number | Publication Date |
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| CN104293644A true CN104293644A (en) | 2015-01-21 |
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| CN201410300855.6A Pending CN104293644A (en) | 2014-06-24 | 2014-06-24 | Assisting part for blood monocyte separation |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567556A (en) * | 2016-01-22 | 2016-05-11 | 武汉海吉力生物科技有限公司 | Density gradient centrifugal tube with position-adjustable porous diaphragm and application of centrifugal tube |
Citations (3)
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| US20020042335A1 (en) * | 2000-04-18 | 2002-04-11 | Anderson Norman G. | Method and apparatus for making density gradients |
| CN101559401A (en) * | 2009-05-26 | 2009-10-21 | 北京化工大学 | Method for separating nano-particles at water-phase density gradient centrifugation rate |
| KR20120123225A (en) * | 2011-04-26 | 2012-11-08 | 서울대학교산학협력단 | Separation Process of Encapsulated Transplantable Cells |
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2014
- 2014-06-24 CN CN201410300855.6A patent/CN104293644A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020042335A1 (en) * | 2000-04-18 | 2002-04-11 | Anderson Norman G. | Method and apparatus for making density gradients |
| CN101559401A (en) * | 2009-05-26 | 2009-10-21 | 北京化工大学 | Method for separating nano-particles at water-phase density gradient centrifugation rate |
| KR20120123225A (en) * | 2011-04-26 | 2012-11-08 | 서울대학교산학협력단 | Separation Process of Encapsulated Transplantable Cells |
Non-Patent Citations (2)
| Title |
|---|
| ZHANG CAI-LONG等: "Isolation of rabbit bone marrow mesenchymal stem cells using density gradient Centrifugation and adherence screening methods", 《中国组织工程研究与临床康》 * |
| 张金花等: "小鼠胰岛密度梯度离心方法的研究", 《浙江医学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567556A (en) * | 2016-01-22 | 2016-05-11 | 武汉海吉力生物科技有限公司 | Density gradient centrifugal tube with position-adjustable porous diaphragm and application of centrifugal tube |
| CN105567556B (en) * | 2016-01-22 | 2017-11-03 | 武汉海吉力生物科技有限公司 | A kind of density gradient centrifugation pipe with the adjustable porous septum in position and its application |
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