CN102329774A - Method for isolating monocytes in bovine peripheral blood - Google Patents
Method for isolating monocytes in bovine peripheral blood Download PDFInfo
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- CN102329774A CN102329774A CN 201110324701 CN201110324701A CN102329774A CN 102329774 A CN102329774 A CN 102329774A CN 201110324701 CN201110324701 CN 201110324701 CN 201110324701 A CN201110324701 A CN 201110324701A CN 102329774 A CN102329774 A CN 102329774A
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Abstract
The invention provides a method for optimally isolating monocytes in bovine peripheral blood. ACD-A is taken as an anticoagulant, the method comprises the optimization of centrifugal conditions, the optimization of cell cleaning conditions, the optimization of wall-sticking conditions and the like, and the success rate of the isolation is greatly improved. In the method, a whole set of complete system which comprises the sampling of bovine blood, the isolation of the monocytes in the bovine peripheral blood and the final transformation of the monocytes into macrophages is established for the first time, and the counting and dyeing are not required in the process, so that the time is saved and an isolation effect is improved. The survival rate of the monocytes isolated by the method is high; and the method is high in experimental repeatability and strong in operability, and provides powerful technical support for simply, conveniently and efficiently isolating the monocytes in the bovine peripheral blood.
Description
Technical field
The present invention relates to cell separation technology, specifically, relate to a kind of method of separating the ox PMBC.
Background technology
Separating periphery blood monocytic cell generally comprises two steps: i.e. separation of PMNC and follow-up monocytic the separation.Wherein, The first step is accomplished through density gradient centrifugation, and through selecting the parting liquid of certain density, (the two density is close for monocyte that can density in the blood is lower and lymphocyte; Close the title PMNC, PBMC) separate with higher granulocyte, the red blood cell component of density; In second step, monocyte can be separated with some other method and lymphocyte, comprises absorption method, immune sorting, reverse elutriation centrifuging and density gradient separation etc.Wherein, absorption method is the most frequently used method, and it is simple, economical, but also has some shortcomings, such as the lymphocyte contamination level is high, handiness is low, workload is big, monocyte activation etc.The immunity sorting is for daily use and handle for a large amount of blood samples very expensively, needs special monoclonal antibody and equipment, generally comprises two kinds of methods of flow cytometry and immunomagnetic beads.Reverse elutriation centrifuging can be handled a large amount of blood, and the high motility rate of purity is high, and is difficult for the activated mononuclear cell, but needs expensive equipment and technical professional.The method of density separation comprises uses two kinds of continuous density gradient and discontinuous density gradients, and the pump and the ultracentrifuge price of wherein making continuous density gradient are relatively costly.
1999; People such as Jindrich Soltys have used the method for immunomagnetic beads to separate the neutrophil leucocyte of ox; Cell yield is higher, purity is purer; And in many detection indexs, all shown better activity, but separate the ox PMBC bibliographical information is not arranged also with immunomagnetic beads method.Use the monocyte stripping technique of antibody to also have flow cytometry, but also be not used in the bibliographical information that separates the ox PMBC.Reverse centrifugal elutriation method needs the special reverse centrifugal elutriation equipment of a cover, and the PMBC that is used for the people separates, but does not also have document to use the PMBC of this technical point from ox.The method of density separation is based on monocyte and lymphocytic varying in size, and uses continuous density gradient parting liquid and ultracentrifuge that the two is separated, this method PMBC separation of ox that also is of little use at present.
Summary of the invention
The present invention is intended to overcome the deficiency of prior art, the plastics absorption method is separated the committed step of ox PMBC and improves, and a kind of method of separation ox PMBC of optimization is provided.
In order to realize the object of the invention, a kind of method of separating the ox PMBC of the present invention comprises step: 1) in the centrifuge tube that contains antithrombotics ACD-A, add the ox peripheral blood that collects, process anticoagulated blood; 2) with OptiPrep
TMStoste and C liquid proportional mixing are processed density separation liquid; 3) with 1) anticoagulated blood and PBS liquid proportional mixing, process the anticoagulated blood of dilution; 4) with 2 parts of volumes 3) anticoagulated blood of dilution be added in 1 part of volume 2) density separation liquid on, centrifugal, abandon supernatant, the white thin layer of parting liquid and serum solution face intersection is transferred in another centrifuge tube; 5) to 4) centrifuge tube in add PBS solution and clean cell, then with the centrifugal 5-10min of rotating speed 250g; Repeat 5) once; 6) to 5) cell precipitation in to add PBS solution resuspended, promptly get mononuclearcell.
Wherein, aforementioned 2) and 3) in the preparation method of C liquid be: 1.79g Tricine Buffer is added in the 100ml water, makes 100mM Tricine storage liquid, 4 ℃ of preservations; 0.85g NaCl is added in the 50ml water, add 20ml 100mM Tricine storage liquid, regulate pH value 7.4, add water and replenish volume to 100ml, 0.22 μ m filter filtration sterilization with 1M NaOH.
Abovementioned steps 1) preparation method of the antithrombotics ACD-A described in is: in 100ml water, add 0.73g Citric Acid, usp, Anhydrous Powder, 2.20g Sodium Citrate, usp, Dihydrate Powder and 2.45g one water Vadex, regulate the pH value to 7.0-7.3,0.22 μ m filter filtration sterilization.Wherein, the volume ratio of antithrombotics ACD-A and ox peripheral blood is 1: 5-10.
Abovementioned steps 2) OptiPrep in
TMThe volume ratio of stoste and C liquid is 1.4: 4.6.
Abovementioned steps 3) volume ratio of anticoagulated blood and PBS liquid is 1: 1 in.
Aforesaid method is further comprising the steps of: with 6) cell suspension be transferred in the Tissue Culture Flask, add the RPMI1640 substratum, then Tissue Culture Flask is placed 37 ℃, 5%CO
2Condition under cultivate, obtain adherent monocyte.
The present invention has adopted novel density gradient separation liquid OptiPrep
TM, it is that a kind of non-ionic type etc. oozes contrast medium, the density range that can dispose is wide, and it is unlike Percoll
TMContain solid granulates, the latter may be engulfed by monocyte, and very low (the high density Ficoll of endotoxin content
TMPossibly contain higher endotoxin content), very little to the monocyte damage and the influence of sensitivity.The present invention is with OptiPrep
TMSpecification sheets is the basis, with reference to other document, design with constantly groping through experiment repeatedly and to obtain about ox and people's monocyte stripping technique, with in specification sheets and the part document to testing favourable step reservation, reject disadvantageous step.
(1) the antithrombotics ACD-A of the present invention's employing can more stably be separated to the better cell of form than other antithrombotics (like heparin and EDTA2K) of routine use; And the suitable long period storing blood of this antithrombotics, the blood of preserving at ambient temperature one day still can be separated to suitable cell.
(2) optimization of cell cleaning condition: the method according to existing document and specification sheets often causes the isolating failure of monocyte; One of reason possibly be the cleaning process of cell; The present invention adopts PBS solution pair cell to clean; Reduced follow-up adherent mononuclear cells is separated adverse factors, for example serum and hematoblastic influence finally makes isolating success ratio increase greatly.
(3) the present invention will sample to separate the ox PMBC and finally be converted into scavenger cell first and form a whole set of complete system from ox blood, and need not counting and dye in the centre, has saved the time and improved separating effect.
(4) adopt the inventive method isolated monocyte survival rate from the ox peripheral blood sample high; Adherent monocyte motility rate is greater than 95%; Experimental repeatability is good, and is workable, and the monocyte that separates in the ox peripheral blood for simple and effective ground provides strong technical support.
Description of drawings
Fig. 1 separates the ox PMBC as antithrombotics, the observed result of microscopically (magnification 200 *) with Fig. 2 for using EDTA.
Fig. 3 separates the ox PMBC as antithrombotics, the observed result of microscopically (magnification 200 *) with Fig. 4 for using heparin.
Fig. 5 separates the ox PMBC as antithrombotics, the observed result of microscopically (magnification 200 *) with Fig. 6 for using ACD-A.
Fig. 7 demonstrate since adherent area too small, monocyte under a large amount of lymphocytic influences, result that can't be adherent fully (magnification 200 *).
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment, the raw materials used commercial goods that is.
Observation of cell uses 20 times of object lens of Olympus IX71 inverted microscope in following examples, and American I magePLUS image analysis system is gathered digital image.
The monocyte that embodiment 1 separates in the ox peripheral blood sample
1, material and reagent
1) antithrombotics ACD-A, compound method is: add 0.73g Citric Acid, usp, Anhydrous Powder, 2.20g Sodium Citrate, usp, Dihydrate Powder and 2.45g one water Vadex in every 100ml water.Regulate the pH value to 7.0-7.3,0.22 μ m filter filtration sterilization.The blood of 1 part of volume ACD-A anti-freezing 5-10 part volume.
2) OptiPrep
TMDensity gradient separation liquid (Axis-Shield PoC, Norway).
3)Tricine?Buffer(Amresco)。
4) the RPMI1640 substratum (Gibco, invitrogen).
5) foetal calf serum (Gibco, invitrogen).
6) PBS (NaCl 8.00g, KCl 0.20g, Na
2HPO
412H
2O 2.91g, KH
2PO
40.24g, add the 800ml deionized water, regulating pH is 7.4, is settled to 1000ml, autoclaving).
2, method
1) aseptic collection ox peripheral blood uses ACD-A antithrombotics (the 50ml centrifuge tube adds about 7ml ACD-A, gathers blood to the 45ml scale marks).
2) according to OptiPrep
TMSpecification sheets (Application Sheet C08), preparation work liquid C liquid.Concrete grammar is: 1.79g Tricine Buffer is added in the 100ml water, make the 100mMTricine storage liquid, 4 ℃ of preservations; 0.85g NaCl is added in the 50ml water, add 20ml100mM Tricine storage liquid, regulate pH value 7.4, add water and replenish volume to 100ml, 0.22 μ m filter filtration sterilization with 1M NaOH.
3) to specifications, with OptiPrep
TMStoste (A liquid) and the mixed of C liquid by 1.4: 4.6 are prepared into 1.068g/ml density separation liquid.
4) with 1) anticoagulated blood and PBS liquid according to 1: 1 mixed.
5) with 2 parts of volumes 4) dilute blood be added to (generally being that 30ml mixing blood is added on the 15ml parting liquid in the 50ml centrifuge tube) on 1 part of volume 1.068g/ml density separation liquid carefully.Pasteur pipet is adopted in suggestion; The centrifuge tube that will contain density separation liquid is inclined to horizontal table top and is 20 degree angles; Pasting tube wall is then slowly adding apart from density separation liquid upper limb 1cm place; Can reduce the disturbance of liquid level, along with the adding of liquid, slowly uprightly (this method sees OptiPrep for details with centrifuge tube
TMSpecification sheets Application Sheet 02).
6) centrifuge tube is placed horizontal rotor room temperature whizzer, centrifugal 30min under the rotating speed of 800g must adopt minimum braking during deceleration.
7) take out centrifuge tube; Supernatant is discarded a part, stay the auxiliary PBMC (the 50ml centrifuge tube approximately stays 5-10ml) of absorption of a part, draw the white thin layer of transparent parting liquid and serum solution face intersection then carefully; Because cell sticks on the centrifugal tube wall easily, should note during absorption.
8) will inhale cell place 15ml centrifuge tube (generally drawing 3-4ml) with 1000 μ L pipettors, add room temperature PBS to 15ml, softly put upside down mixing 5 times, place horizontal rotor room temperature whizzer then, centrifugal 7min under the 250g rotating speed adopts semi-brake.Employing is cleaned cell one time again with quadrat method, adopts full application of brake when centrifugal.
9) cell in each 15ml centrifuge tube is resuspended fully softly with 0.5ml PBS, add a 25cm
2In the plastics Tissue Culture Flask, in culturing bottle, add 5.5ml room temperature RPMI1640 perfect medium (containing 10% foetal calf serum, 1% green grass or young crops/Streptomycin sulphate) then, the mixing that slightly vibrates makes cell be tiled in evenly that (this moment, cell density was about 2.5 * 10 in the culturing bottle
6Individual/ml).With the cell bottle under 37 ℃, 5%CO
2Condition under cultivate.Promptly get the PBMC cell, can be used for subsequent experimental.
10) cultivate after 1 day, can remove upper strata enchylema, and embathe twice, add perfect medium then, continue to cultivate adherent cell with room temperature PBS.To the 5th day, adherent monocyte just can be converted into scavenger cell, can be used for the downstream experiment.
Embodiment 2 antithrombotics are to separating the influence of ox PMBC
The present invention improves details step and condition on the basis of conventional separation method.At first be the selection of antithrombotics, secondly preferred ACD-A is heparin, does not advise using EDTA.Then, thrombocyte, residual serum etc. must be fully removed in the cleaning of cell.At last, formed the scheme of a cover medelling, comprised that use 50ml centrifuge tube is centrifugal, the 15ml centrifuge tube cleans, 25cm
2The scheme that culturing bottle is adherent has good repeatability and operability, does not need cell counting, saves experimental period, improves separating effect.
The present invention has groped the working conditions of antithrombotics, comprises heparin (2mg/10ml blood), EDTA dipotassium (K
2EDTA, 18mg/10ml blood) and ACD-A.The result finds, the PBMC quantity that three kinds of antithrombotics obtain has notable difference.
Gather the blood sample of two cow head I and I ' respectively, by the same operator according to OptiPrep
TMMethod described in the specification sheets is separated simultaneously.The PBMC cell that obtains takes a morsel after resuspended with substratum and to dye remaining being incubated in the cell bottle with trypan blue.After the trypan blue dyeing, count and classify through the cell counter pair cell.(table 1)
Isolate cell motility rate and the quantity of PBMC behind the different antithrombotics anticoagulated bloods of table 1
Can find out that from table 1 there were significant differences to use different antithrombotics to separate the PBMC quantity that obtains.K
2Significantly more than other two kinds of anticoagulations, ACD-A and heparin are respectively the former 44% and 69% to the PBMC quantity that the EDTA anticoagulation is separated to.If consider K
2EDTA is a dry powder form, can be not that liquid form can dilute blood as heparin and ACD-A outside (in the 50ml centrifuge tube, TV is 45ml, and ACD-A and heparin add 7ml and 5ml respectively, and the dilution of blood is respectively 84% and 89%), K
2The EDTA antithrombotics from the unit volume blood separation to PBMC quantity still significantly more than other two kinds of antithrombotics (ACD-A and heparin are respectively its 52% and 78%).In fact, when after density gradient centrifugation, from whizzer, taking out centrifuge tube, can directly find out K
2The cellular layer at the interface in the EDTA pipe than two other antithrombotics thickness of pipe many.The effect of this antithrombotics and the classification of parting liquid are irrelevant, and the present invention has also used Ficoll
TMDensity gradient separation liquid has been tested three antithrombotics, has also obtained similar effects.
But the pertinent literature report is arranged, and EDTA possibly cause thrombocyte tightly to be adsorbed in monocyte, possibly cause the latter to discharge some cytokines.And the cell that is separated to of EDTA sometimes, observed cellular form is all not ideal enough in follow-up cultivation, breaks up less (Fig. 1 and Fig. 2).
Other has report, and heparin may cause platelet aggregation, and activates thrombocyte, and thrombocyte just in time is in the PBMC layer, so may impact PBMC.But also there are some documents to use heparin to come separating monocytic cell as antithrombotics.Rule of thumb, use the heparin effect comparatively stable, the cell quantity that is separated to is as shown in table 1, is in medium.Also better on the form, the situation (Fig. 3 and Fig. 4) of some differentiation has appearred.
Adopt ACD-A as antithrombotics, though the PBMC cell quantity that obtains is minimum, the cellular form after its separation is (Fig. 5 and Fig. 6) better.
Carried out twice experiment later on again and groped, the separating effect that can find out ACD-A is more stable.Rule of thumb; Clean a large amount of situation about reducing of back cell quantity owing to use EDTA to occur in easily; And cellular form is not as the separating effect of heparin and ACD, and has document to think that it can cause platelet adhesion in monocyte and the latter is activated, so do not advise use.
Embodiment 3 cells clean separating the influence of ox PMBC
The present invention except done in the selection of antithrombotics grope, also the treatment process of the cell that is separated to is groped.Wherein the most key step is exactly that pair cell cleans fully.In experiment before, such situation often occurs: separating step is normal fully, and before adding substratum, mirror disperses between the inspection cell down each other.But adding perfect medium when adherent, the rapid phase mutual coagulation of cell, the cell condensation in last whole hole becomes a macroscopic film, and it is few to remove attached cell quantity remaining behind the film.Because the cleaning of pair cell only is to operate according to the specification sheets of parting liquid, directly adds behind the parting liquid the centrifugal cell that obtains and mix with the C liquid (about 8ml) of two volumes that also 500g is centrifugal.Adding 5ml PBS again cleans 1 time.Yet this cleaning still can not be removed compositions such as serum and the thrombocyte of original blood fully, and this like cell activates rapidly when the contact perfect medium easily, assembles each other.So the present invention has strengthened the amount of cleaning, all in the 15ml centrifuge tube, filled it up with PBS during twice cleaning.What need pay special attention in addition is; After the 15ml centrifuge tube is each centrifugal, can not directly add PBS or substratum, (amount of liquid can not be too much to add 1ml PBS or substratum earlier; Otherwise be difficult to blow even); Fully add remaining volume again after the re-suspended cell lightly with 1000 μ l rifle heads, otherwise cell still can be in mutual accumulative state, influences follow-up adherent effect.For OptiPrep
TMThe high RCF rotating speed of 500g is used in suggestion in the specification sheets, and it also is not suitable for plastics absorption method separation ox monocyte, because monocyte is easy to sedimentation, it is resuspended that higher centrifugal speed is difficult for cell, so the present invention adopts the centrifugal 5-10min of 250g.For separating monocytic cell, cf-and centrifugation time during cleaning can be still less.
Embodiment 4 plastics absorption methods obtain the medelling program of the scavenger cell of cells of monocytic origin
Often to measure before adherent through trypan blue dyeing carrying out cell counting and cell motility rate; Use PBMC if desired; Can carry out this step operation; But should earlier the remainder cell that adds perfect medium be added in the culturing bottle, place incubator to cultivate, and then carry out counting operation.Use adherent monocyte if desired; Rule of thumb need not count; Since difficult differentiation of this moment monocyte and lymphocyte, and form is similar under the mirror, and the count results of gained does not have practical significance; But can be used to judge the quantity of the cell that is separated to, adjustment follow-up test scheme for the laboratory of isolated cell first.For the cell motility rate, after trypan blue dyeing, find that the attached cell that is cultured to the 5th day nearly all survives; Because pair cell dyeing can only be carried out in the cell bottle,, have a negative impact because understand pair cell so the cell that is inappropriate for experimental group and control group dyes; If so conditions permit; Can set up one or two bottle of cell dyeing separately,, directly after being separated to cell, observe form under the mirror and quantity gets final product if there is not condition not dye.
For the density of cell attachment, rule of thumb, add 15ml parting liquid and 30ml mixing blood (containing 15ml blood and 15ml PBS) in the centrifugal preceding 50ml centrifuge tube; 3-4ml is drawn in centrifugal back; After cleaning 2 times, add the 3ml perfect medium, just can transfer to a 25cm
2Tissue Culture Flask, and supply substratum to 6ml, such cell density is comparatively suitable.Use 6 orifice plates to carry out cell attachment cultivates before; Though but found afterwards that 6 orifice plates were easy to use in follow-up test; But because its single hole volume is less, do not have enough adherent areas, all can produce detrimentally affect lymphocyte and monocyte; And 6 orifice plate also be difficult for pair cell and embathe, so adopt 25cm among the present invention
2Tissue Culture Flask.In the experiment of adherent mononuclear cells, exist a contradiction, that is exactly adherent area and the contradiction between the cell density; If adherent area is too small, monocyte can't adherent fully (Fig. 7) under a large amount of lymphocytic influences; If adherent area is excessive; Then the cell density of follow-up test can be very low, and some document uses more large-area petridish (15cm diameter) to carry out cell attachment, with EDTA adherent cell suspension is got up afterwards; Transfer in the less container of the area that needs operation, improve cell density whereby.Rule of thumb, add after the EDTA, the cell after suspending has almost lost adherent ability again, so do not advise using this method.Through groping the 25cm that the present invention uses
2The cell that the culturing bottle adherent culture obtains meets the requirements.Use TRIzol
TM(invitrogen) handled two bottles of monocytes of the 6th day that obtain through this method, the cell total rna amount of extracting all reaches 1.7 μ g, shows that such cell quantity is suitable for general test.
The present invention has adopted OptiPrep
TMParting liquid, this commercial parting liquid have been proved the metabolism and the not influence of vigor of pair cell.The scavenger cell separation that the present invention is directed to the conversion of ox PMBC is optimized; The selection, centrifugal condition, cell cleaning condition, the adherent condition that comprise antithrombotics; Step is detailed; Improve the success ratio of experiment greatly, reduced plenty of time, material and energy cost that experimenter's condition of groping spends.
Concrete advantage show following some:
1, the present invention has adopted novel density gradient separation liquid OptiPrep
TM, it is that a kind of non-ionic type etc. oozes contrast medium, the density range that can dispose is wide, and it is unlike Percoll
TMContain solid granulates, the latter may be engulfed by monocyte, and very low (the high density Ficoll of endotoxin content
TMPossibly contain higher endotoxin content), very little to the monocyte damage and the influence of sensitivity.
2, the antithrombotics ACD-A of the present invention's employing is than other antithrombotics (heparin and the K of routine use
2EDTA) can more stably be separated to the better cell of form, and the suitable long period storing blood of this antithrombotics, the blood of preserving at ambient temperature one day still can be separated to suitable cell.
3, the cleaning process of cell adopts a large amount of PBS to clean cell twice, and reduced follow-up adherent mononuclear cells has been separated adverse factors, for example serum and hematoblastic influence, and adjusted centrifugal condition, the final success ratio of experiment that makes increases greatly.
4, set up first from ox blood and sampled a whole set of complete system of separating the ox PMBC and finally being converted into scavenger cell, used 50ml centrifuge tube, 15ml centrifuge tube and 25cm
2The supporting system of Tissue Culture Flask, need not counting and dye in the centre, has saved the time and improved separating effect.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (7)
1. a method of separating the ox PMBC is characterized in that, comprises step:
1) in the centrifuge tube that contains antithrombotics ACD-A, adds the ox peripheral blood that collects, process anticoagulated blood;
2) with OptiPrep
TMStoste and C liquid proportional mixing are processed density separation liquid;
3) with 1) anticoagulated blood and PBS solution proportional mixing, process the anticoagulated blood of dilution;
4) with 2 parts of volumes 3) anticoagulated blood of dilution be added in 1 part of volume 2) density separation liquid on, centrifugal, abandon supernatant, the white thin layer of parting liquid and serum solution face intersection is transferred in another centrifuge tube;
5) to 4) centrifuge tube in add PBS solution and clean cell, then with the centrifugal 5-10min of rotating speed 250g;
6) to 5) cell precipitation in to add PBS solution resuspended, promptly get mononuclearcell;
The preparation method of the C liquid wherein, 2) is: 1.79g Tricine Buffer is added in the 100ml water, make 100mM Tricine storage liquid, 4 ℃ of preservations; 0.85g NaCl is added in the 50ml water, add 20ml 100mM Tricine storage liquid, regulate pH value 7.4, add water and replenish volume to 100ml, 0.22 μ m filter filtration sterilization with 1M NaOH.
2. method according to claim 1; It is characterized in that; The preparation method of antithrombotics ACD-A described in the step 1) is: in 100ml water, add 0.73g Citric Acid, usp, Anhydrous Powder, 2.20g Sodium Citrate, usp, Dihydrate Powder and 2.45g one water Vadex; Regulate the pH value to 7.0-7.3,0.22 μ m filter filtration sterilization.
3. method according to claim 2 is characterized in that, the volume ratio of antithrombotics ACD-A and ox peripheral blood is 1: 5-10.
4. method according to claim 1 is characterized in that step 2) middle OptiPrep
TMThe volume ratio of stoste and C liquid is 1.4: 4.6.
5. method according to claim 3 is characterized in that, the volume ratio of anticoagulated blood and PBS liquid is 1: 1 in the step 3).
6. method according to claim 1 is characterized in that, between step 5) and step 6), repeats once to clean the step of cell.
7. according to each described method of claim 1-6, it is characterized in that, further comprising the steps of: with 6) cell suspension be transferred in the Tissue Culture Flask, add the RPMI1640 substratum, then Tissue Culture Flask is placed 37 ℃, 5%CO
2Condition under cultivate, obtain adherent monocyte.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108642005A (en) * | 2018-05-22 | 2018-10-12 | 广州迪澳医疗科技有限公司 | A kind of lymphocyte separation medium containing bridging agent |
| CN113106060A (en) * | 2020-02-25 | 2021-07-13 | 河南省银丰生物工程技术有限公司 | PBMC cell in-vitro culture method |
| CN114891745A (en) * | 2022-05-18 | 2022-08-12 | 黑龙江八一农垦大学 | Separation and purification of CD14 in cow blood + Method for producing monocytes |
-
2011
- 2011-10-21 CN CN 201110324701 patent/CN102329774B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
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| 《the scientific world Journal》 20050615 John M. Graham Isolation of Peripheral Blood Mononuclear Cells from Macaques on a Density Barrier 1654-1656 1-7 第2卷, * |
| 《中国奶业协会2009年会论文集》 20091231 郭熠洁等 脂多糖刺激对牛单核细胞由来的巨噬细胞Toll样受体表达的影响 268-271 1-7 , * |
| 《广东医药学院学报》 19921231 吕小迅等 四种抗凝剂对人血单核细胞分离和趋化性的影响 11-13 1-7 第8卷, 第2期 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108642005A (en) * | 2018-05-22 | 2018-10-12 | 广州迪澳医疗科技有限公司 | A kind of lymphocyte separation medium containing bridging agent |
| CN108642005B (en) * | 2018-05-22 | 2021-07-27 | 广州迪澳医疗科技有限公司 | Lymphocyte separation liquid containing connecting agent |
| CN113106060A (en) * | 2020-02-25 | 2021-07-13 | 河南省银丰生物工程技术有限公司 | PBMC cell in-vitro culture method |
| CN114891745A (en) * | 2022-05-18 | 2022-08-12 | 黑龙江八一农垦大学 | Separation and purification of CD14 in cow blood + Method for producing monocytes |
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| Publication number | Publication date |
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| CN102329774B (en) | 2013-01-02 |
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