CN103289953A - Fish leukocyte separation method - Google Patents
Fish leukocyte separation method Download PDFInfo
- Publication number
- CN103289953A CN103289953A CN2013101977298A CN201310197729A CN103289953A CN 103289953 A CN103289953 A CN 103289953A CN 2013101977298 A CN2013101977298 A CN 2013101977298A CN 201310197729 A CN201310197729 A CN 201310197729A CN 103289953 A CN103289953 A CN 103289953A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- white corpuscle
- fish
- separation method
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention provides a fish leukocyte separation method. The method comprises a step of inoculating fish complete blood cells into an animal cell culture medium without animal serum to culture under an aseptic condition, wherein the complete blood cell inoculation intensity is 10<6>-10<10>/milliliter culture medium, and the culture conditions are that the temperature is 15 DEG C-37 DEG C, the humidity is 90%-98% and the content of carbon dioxide is 0-8%. A cell separation liquid or proper cell lysates does not need to be additionally used, the leukocyte is successfully separated through adopting a method of combining a cell culture fluid with temperature, and protein ingredients such as animal serum do not need to be used in the culture medium; the leukocyte separated by utilizing the method does not have side effects generated by chemical reagents such as the separation liquid, the lystate and the like, and has the advantages of high leukocyte purity, strong activity, good safety and low cost; in addition, the leukocyte separated by utilizing the method can normally grow in the culture medium with low serum content in a vitro culture process.
Description
Technical field
The present invention relates to the leukocytic separation method of a kind of animal, particularly a kind of fish white corpuscle separation method that is applicable to vitro culture.
Background technology
White corpuscle has defence and immunologic function, cures, resists the invasion of cause of disease at body injury and the immunology of disease is played an important role.Study the immunologic mechanism of body, obtaining sufficient amount, purity height and active strong white corpuscle is primary condition, and the foundation of leukocytic vitro culture and stimulus modality is prerequisite.Existing white corpuscle separation method mainly contains two kinds: a kind of is to adopt the direct splitting erythrocyte of erythrocyte cracked liquid and separate white corpuscle, and another kind is to adopt the lymph parting liquid to carry out gradient centrifugation and separate each hemocyte component.The white corpuscle purity height of separating with first method, but poor activity is not suitable for further carrying out vitro culture, and therefore the white corpuscle with this method separation is mainly used in leukocytic temporary detecting; Better with the white corpuscle vigor that second method is separated, but component is more single, have only lymphocyte mostly, and the granulocyte close with red corpuscle density is dropped, and when vitro culture, need add relevant cofactor.
Summary of the invention
In order to overcome the deficiency that above-mentioned prior art exists, the invention provides a kind of brand-new fish white corpuscle separation method, this method uses the Zooblast culture medium that does not contain serum just white corpuscle successfully can be separated with red corpuscle, and the isolated white corpuscle of institute is active high, is easy to vitro culture.
Technical scheme of the present invention provides a kind of fish white corpuscle separation method, and this method is under aseptic condition, the fish complete blood cell is inoculated in the Zooblast culture medium that does not contain animal serum cultivates, and wherein the complete blood cell inoculum density is 10
6-10
10Individual/the milliliter substratum, culture condition is 15 ℃-37 ℃ temperature, the humidity of 90%-98% and the carbonic acid gas of 0-8 %.
Described complete blood cell inoculum density is preferably 10
7~ 10
9Individual/the milliliter substratum, the complete blood cell inoculum density is in this scope, and the net result difference that white corpuscle separates is little.
Described culture condition is preferably 25 ℃-30 ℃ temperature, the humidity of 92%-96% and the carbonic acid gas of 3-5 %.
The described Zooblast culture medium that does not contain animal serum is made up of culture medium dry powder composition and supporting agent, and wherein the component of culture medium dry powder composition and content are that every liter of substratum comprises Calcium Chloride Powder Anhydrous .2H2O 250.00-270.00 mg, iron nitrate .9H2O 0.05-0.15 mg, Repone K 300.00-500.00 mg, anhydrous magnesium sulfate 95.00-100.00 mg, sodium-chlor 6300.00-6500.00 mg, AMSP 108.00-110.00 mg, Succinic Acid 70.00-80.00 mg, Soduxin 90.00-110.00 mg, L-arginine hydrochloride 82.00-86.00 mg, L-hydrochloric acid Gelucystine 60.00-65.00 mg, glycine 20.00-40.00 mg, L-histidine monohydrochloride 40.00-44.00 mg, L-Isoleucine 100.00-110.00 mg, L-leucine 100.00-110.00 mg, L-lysine hydrochloride 144.00-148.00 mg, L-methionine(Met) 20.00-60.00 mg, L-phenylalanine 64.00-68.00 mg, L-Serine 38.00-45.00 mg, L-Threonine 90.00-100.00 mg, L-tryptophane 14.00-18.00 mg, L-tyrosine 70.00-74.00 mg, L-Xie Ansuan 92.00-96.00 mg, D-calcium pantothenate 2.00-6.00 mg, choline tartrate 7.00-7.40 mg, folic acid 2.00-6.00 mg, inositol 7.00-7.60 mg, niacinamide 2.00-6.00 mg, riboflavin 0.20-0.60 mg, thiamine hydrochloride 2.00-6.00 mg, pyridoxine hydrochloride 2.00-6.00 mg, glucose 800.00-3000.00 mg, Sodium.alpha.-ketopropionate 100.00-120.00 mg, phenol red 9.00-9.60 mg, sodium bicarbonate 3500.00-4000.00 mg.
The component of described culture medium dry powder composition and content are preferably every liter of substratum and comprise Calcium Chloride Powder Anhydrous .2H2O 255.00-265.00 mg, iron nitrate .9H2O 0.08-0.12 mg, Repone K 350.00-450.00 mg, anhydrous magnesium sulfate 96.00-98.00 mg, sodium-chlor 6350.00-6450.00 mg, AMSP 108.00-110.00 mg, Succinic Acid 72.00-78.00 mg, Soduxin 95.00-105.00 mg, L-arginine hydrochloride 83.00-85.00 mg, L-hydrochloric acid Gelucystine 62.00-64.00 mg, glycine 25.00-35.00 mg, L-histidine monohydrochloride 41.00-43.00 mg, L-Isoleucine 101.00-109.00 mg, L-leucine 101.00-109.00 mg, L-lysine hydrochloride 145.00-147.00 mg, L-methionine(Met) 205.00-55.00 mg, L-phenylalanine 65.00-67.00 mg, L-Serine 40.00-44.00 mg, L-Threonine 92.00-98.00 mg, L-tryptophane 15.00-17.00 mg, L-tyrosine 71.00-73.00 mg, L-Xie Ansuan 93.00-95.00 mg, D-calcium pantothenate 2.50-5.50 mg, choline tartrate 7.10-7.30 mg, folic acid 3.00-5.00 mg, inositol 7.10-7.50 mg, niacinamide 3.00-5.00 mg, riboflavin 0.30-0.50 mg, thiamine hydrochloride 3.00-5.00 mg, pyridoxine hydrochloride 3.00-5.00 mg, glucose 900.00-2000.00 mg, Sodium.alpha.-ketopropionate 105.00-115.00 mg, phenol red 9.10-9.50 mg, sodium bicarbonate 3600.00-3800.00 mg.
Each component concentration of culture medium dry powder composition in the above-mentioned Zooblast culture medium that does not contain animal serum within the specific limits, the net result that its white corpuscle separates is similar.
One of described Zooblast culture medium constituent supporting agent is preferably one or more in distilled water, deionized water, water for injection, redistilled water and the ultrapure water.
The preparation method of described Zooblast culture medium is dispersed in Zooblast culture medium dry powder composition in the supporting agent, refilters degerming.
The dispersing method of described Zooblast culture medium dry powder composition in supporting agent can be realized by this area dispersing method commonly used, as sonic oscillation, magnetic agitation etc.; Described filtration sterilization method is that bacteriological filtration film well known in the art carries out the one or many filtration, and wherein the aperture of bacteriological filtration film is not more than 0.22 μ m.
The all ingredients that the present invention uses can be purchased, and also can adopt this area method preparation commonly used.The reagent that is used for cell cultures, purity requirement height not only, and can not have any material that influences the cell normal physiological activity, therefore also need to meet the requirement of pharmaceutical products, preferred injection stage raw material reagent.
White corpuscle separation method of the present invention can separate freshwater fish white corpuscles such as swamp eel white corpuscle, grass carp white corpuscle and crucian, and the white corpuscle after the separation can carry out vitro culture.
Advantage of the present invention is: the invention provides a kind of brand-new fish white corpuscle separation method, need not additionally to use the lysate of cellular segregation liquid or certain cell, but the method that combines by cell culture fluid and temperature, white corpuscle is successfully separated, also need not to use the protein ingredient of animal serum and so in the substratum; The inventive method institute isolated cells, the side effect that does not have the chemical reagent of parting liquid and lysate and so on to produce, the cell purity height, active strong, security is good, and cost is low; In addition, the cell of separating with the inventive method, when carrying out vitro culture, but in the low levels blood serum medium normal growth just, use the inventive method isolated cells, different batches difference is little.Therefore the present invention has simplified the leukocytic isolation technique of fish, saves cost, improves the white corpuscle vigor.
Description of drawings
The white corpuscle photo that Fig. 1 separates for the white corpuscle separation method of embodiment 1;
The white corpuscle photo that Fig. 2 separates for the white corpuscle separation method of embodiment 2;
The white corpuscle photo that Fig. 3 separates for the white corpuscle separation method of Comparative Examples 1;
The white corpuscle photo that Fig. 4 separates for the white corpuscle separation method of Comparative Examples 2;
Fig. 5 cultivates 72 hours picture for the white corpuscle that the white corpuscle separation method of embodiment 1 is separated;
Fig. 6 cultivates 72 hours picture for the white corpuscle that the white corpuscle separation method of embodiment 2 is separated;
Fig. 7 cultivates 72 hours picture for the white corpuscle that the white corpuscle separation method of Comparative Examples 1 is separated;
Fig. 8 cultivates 72 hours picture for the white corpuscle that the white corpuscle separation method of Comparative Examples 2 is separated.
Embodiment
Now in conjunction with specific embodiments, technology contents of the present invention is further described.
This embodiment of embodiment 1(is used for explanation white corpuscle separation method of the present invention)
The used substratum of present embodiment does not contain any animal serum, and each component of described culture medium dry powder composition and content are as shown in table 1:
Table 1
The mg/L of unit of each component of culture medium dry powder composition refers in table 1 and following each table, be benchmark with the volume with this culture medium dry powder composition and the final liquid nutrient medium of carrier mixing gained, be equivalent to every liter of final liquid nutrient medium, the milligram number of the solid matter that when the preparation substratum, need add.
The concrete preparation method of the Zooblast culture medium that the present invention is used is with the consumption of each component except sodium bicarbonate of the solid medium dry powder composite shown in the table 1 according to 1 liter of final liquid nutrient medium, after the one accurate weighing mixing of ten thousand/balance, add 900 milliliters of ultrapure waters again, obtain uniform dispersion 25 ℃ of stirrings after 2 hours with magnetic stirring apparatus, add 3.7 gram sodium bicarbonates then its pH value is transferred to 7.0, be settled to 1000 milliliters with ultrapure water, with the bacteriological filtration membrane filtration degerming of 0.22 μ m, 4 ℃ of preservations are standby behind the constant volume.
Leukocytic concrete separation method is under aseptic condition, with pipettor the above-mentioned nutrient solution of 5ml is transferred to 25cm
2In the Tissue Culture Flask, inoculate fresh swamp eel complete blood cell, inoculum density is 2 * 10
7Individual cell/ml substratum; Under 25 ℃ of temperature condition, 95% humidity condition and 5% carbon dioxide conditions, cultivated 3-7 days then, the time that record unit odd number red corpuscle and leukocytic relative proportion and red corpuscle all disappear under the inverted microscope, the Rui Shi Giemsa staining detects the cell of institute's culturing cell and forms.The Rui Shi Giemsa staining the results are shown in Figure 1, and 72 hours photo of cell cultures is seen Fig. 5.As seen from Figure 1,25 ℃ of complete blood cell of cultivating 72 hours down, mainly formed by lymphocyte, monocyte, granulocyte, do not had red corpuscle.As seen from Figure 5, the complete blood cell of being cultivated is after 72 hours, and red corpuscle all disappears, and remaining white corpuscle is in good condition.
Embodiment 2 (this embodiment is used for explanation white corpuscle separation method of the present invention)
The used substratum of present embodiment does not contain any animal serum, and each component of described culture medium dry powder composition and content are with table 1.
The preparation method of the used Zooblast culture medium of the present invention such as embodiment 1, and leukocytic concrete separation method is under aseptic condition, with pipettor the above-mentioned nutrient solution of 5ml is transferred to 25cm
2In the Tissue Culture Flask, inoculate fresh swamp eel complete blood cell, inoculum density is 2 * 10
7Cell/ml; Under 37 ℃ of temperature condition, 95% humidity condition and 5% carbon dioxide conditions, cultivated 2-4 days then, the time that record unit odd number red corpuscle and leukocytic relative proportion and red corpuscle all disappear under the inverted microscope, the Rui Shi Giemsa staining detects the cell of institute's culturing cell and forms.The Rui Shi Giemsa staining the results are shown in Figure 2, and 72 hours photo of cell cultures is seen Fig. 6.As seen from Figure 2, in the complete blood cell of 37 ℃ of cultivations after 72 hours, mainly formed by granulocyte, lymphocyte, monocyte, do not had red corpuscle.As seen from Figure 6, after 72 hours, red corpuscle all disappears in complete blood cell that 37 ℃ of quilts are cultivated, and remaining white corpuscle is in good condition.
Below see two groups of contrast experiment's examples, they are used for the existing white corpuscle separation method of explanation.
Comparative Examples 1
The erythrocyte splitting fluid component is as shown in the table:
Under aseptic condition, with pipettor the above-mentioned lysate of 2ml is transferred in the 10ml centrifuge tube, add the fresh finless eel blood of 2ml then, room temperature left standstill 10 minutes behind the mixing, after treating that solution is transparent, it is under the 1500rpm condition centrifugal 5 minutes at 25 ℃ of temperature condition, rotating speed, abandon supernatant, after phosphoric acid buffer is resuspended with cell precipitation, rotating speed is under the 1500rpm condition centrifugal 5 minutes, abandon supernatant, with embodiment 1 used same medium re-suspended cell, under embodiment 1 same culture conditions, cultivate a washing back part and transfer to 25cm with pipettor
2In the Tissue Culture Flask, use the substratum identical with embodiment 1, by cultivating under the identical condition of embodiment 1, do the Rui Shi Giemsa staining simultaneously and detect cell and form, cultured cells observation of cell form and taking pictures under inverted microscope.Hemocyte behind the erythrocyte splitting is formed the Rui Shi Giemsa staining and be the results are shown in Figure 3.The state that hemocyte behind the erythrocyte splitting was cultivated after 72 hours is seen Fig. 7.As seen from Figure 3, remove red corpuscle with conventional cracking process after, mainly by mainly being made up of macrolymphocyte, small lymphocyte, monocyte, granulocyte, it forms identical with the white corpuscle composition of separating with the present invention remaining cell.As seen from Figure 7, handle white corpuscle afterwards with erythrocyte cracked liquid, under non-additive situation, cultivate, dead by cultured cells most of cell after 72 hours.
Comparative Examples 2
The fish lymphocyte separation medium is available from Tianjin Hao ocean biological products company limited.Under aseptic condition, with pipettor the above-mentioned lymph parting liquid of 2ml is transferred in the 10ml centrifuge tube, adding the fresh finless eel blood of 2ml along tube wall then, is under the 2000rpm condition centrifugal 15 minutes at 25 ℃ of temperature condition, rotating speed, transfers to 25cm after getting the cell phosphoric acid buffer washing of buffy coat
2In the Tissue Culture Flask, with the identical substratum of embodiment 1, by cultivating under the identical condition of embodiment 1, do the Rui Shi Giemsa staining simultaneously and detect cell and form.Observe the culturing cell form under the inverted microscope and take pictures.The cell Rui Shi Giemsa staining that the lymph parting liquid is separated the results are shown in Figure 4.72 hours state of cell cultures is seen Fig. 8, as seen from Figure 4, with the lymph confluent monolayer cells that conventional density gradient centrifugation is separated, mainly is made up of macrolymphocyte, small lymphocyte.As seen from Figure 8, with the lymphocyte that the lymph parting liquid is separated by density gradient centrifugation, under non-additive situation, cultivate, begun death by cultured cells most of cell after 72 hours.
Claims (7)
1. fish white corpuscle separation method, it is characterized in that: this method is under aseptic condition, the fish complete blood cell is inoculated in the Zooblast culture medium that does not contain animal serum cultivates, wherein the complete blood cell inoculum density is 10
6-10
10Individual/the milliliter substratum, culture condition is 15 ℃-37 ℃ temperature, the humidity of 90%-98% and the carbonic acid gas of 0-8 %.
2. fish white corpuscle separation method according to claim 1, it is characterized in that: described complete blood cell inoculum density is preferably 10
7~ 10
9Individual/the milliliter substratum.
3. fish white corpuscle separation method according to claim 2, it is characterized in that: described culture condition is preferably 25 ℃-30 ℃ temperature, the humidity of 92%-96% and the carbonic acid gas of 3-5 %.
4. according to claim 1 or 2 or 3 described fish white corpuscle separation methods, it is characterized in that: one of described Zooblast culture medium constituent supporting agent is preferably one or more in distilled water, deionized water, water for injection, redistilled water and the ultrapure water.
5. fish white corpuscle separation method according to claim 4, it is characterized in that: the preparation method of described Zooblast culture medium is dispersed in Zooblast culture medium dry powder composition in the supporting agent, refilters degerming.
6. fish white corpuscle separation method according to claim 5, it is characterized in that: the described Zooblast culture medium that does not contain animal serum is made up of culture medium dry powder composition and supporting agent, and wherein the component of culture medium dry powder composition and content are that every liter of substratum comprises Calcium Chloride Powder Anhydrous .2H2O 250.00-270.00 mg, iron nitrate .9H2O 0.05-0.15 mg, Repone K 300.00-500.00 mg, anhydrous magnesium sulfate 95.00-100.00 mg, sodium-chlor 6300.00-6500.00 mg, AMSP 108.00-110.00 mg, Succinic Acid 70.00-80.00 mg, Soduxin 90.00-110.00 mg, L-arginine hydrochloride 82.00-86.00 mg, L-hydrochloric acid Gelucystine 60.00-65.00 mg, glycine 20.00-40.00 mg, L-histidine monohydrochloride 40.00-44.00 mg, L-Isoleucine 100.00-110.00 mg, L-leucine 100.00-110.00 mg, L-lysine hydrochloride 144.00-148.00 mg, L-methionine(Met) 20.00-60.00 mg, L-phenylalanine 64.00-68.00 mg, L-Serine 38.00-45.00 mg, L-Threonine 90.00-100.00 mg, L-tryptophane 14.00-18.00 mg, L-tyrosine 70.00-74.00 mg, L-Xie Ansuan 92.00-96.00 mg, D-calcium pantothenate 2.00-6.00 mg, choline tartrate 7.00-7.40 mg, folic acid 2.00-6.00 mg, inositol 7.00-7.60 mg, niacinamide 2.00-6.00 mg, riboflavin 0.20-0.60 mg, thiamine hydrochloride 2.00-6.00 mg, pyridoxine hydrochloride 2.00-6.00 mg, glucose 800.00-3000.00 mg, Sodium.alpha.-ketopropionate 100.00-120.00 mg, phenol red 9.00-9.60 mg, sodium bicarbonate 3500.00-4000.00 mg.
7. fish white corpuscle separation method according to claim 5, it is characterized in that: the component of described culture medium dry powder composition and content are preferably every liter of substratum and comprise Calcium Chloride Powder Anhydrous .2H2O 255.00-265.00 mg, iron nitrate .9H2O 0.08-0.12 mg, Repone K 350.00-450.00 mg, anhydrous magnesium sulfate 96.00-98.00 mg, sodium-chlor 6350.00-6450.00 mg, AMSP 108.00-110.00 mg, Succinic Acid 72.00-78.00 mg, Soduxin 95.00-105.00 mg, L-arginine hydrochloride 83.00-85.00 mg, L-hydrochloric acid Gelucystine 62.00-64.00 mg, glycine 25.00-35.00 mg, L-histidine monohydrochloride 41.00-43.00 mg, L-Isoleucine 101.00-109.00 mg, L-leucine 101.00-109.00 mg, L-lysine hydrochloride 145.00-147.00 mg, L-methionine(Met) 205.00-55.00 mg, L-phenylalanine 65.00-67.00 mg, L-Serine 40.00-44.00 mg, L-Threonine 92.00-98.00 mg, L-tryptophane 15.00-17.00 mg, L-tyrosine 71.00-73.00 mg, L-Xie Ansuan 93.00-95.00 mg, D-calcium pantothenate 2.50-5.50 mg, choline tartrate 7.10-7.30 mg, folic acid 3.00-5.00 mg, inositol 7.10-7.50 mg, niacinamide 3.00-5.00 mg, riboflavin 0.30-0.50 mg, thiamine hydrochloride 3.00-5.00 mg, pyridoxine hydrochloride 3.00-5.00 mg, glucose 900.00-2000.00 mg, Sodium.alpha.-ketopropionate 105.00-115.00 mg, phenol red 9.10-9.50 mg, sodium bicarbonate 3600.00-3800.00 mg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310197729.8A CN103289953B (en) | 2013-05-24 | 2013-05-24 | Fish leukocyte separation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310197729.8A CN103289953B (en) | 2013-05-24 | 2013-05-24 | Fish leukocyte separation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103289953A true CN103289953A (en) | 2013-09-11 |
| CN103289953B CN103289953B (en) | 2015-10-07 |
Family
ID=49091491
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310197729.8A Expired - Fee Related CN103289953B (en) | 2013-05-24 | 2013-05-24 | Fish leukocyte separation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103289953B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110438075A (en) * | 2019-08-13 | 2019-11-12 | 贵州省水产研究所 | The sturgeon leucocyte separation method of simple and efficient |
-
2013
- 2013-05-24 CN CN201310197729.8A patent/CN103289953B/en not_active Expired - Fee Related
Non-Patent Citations (5)
| Title |
|---|
| J. CHRISTOPHER LUFT, 等: "A serum-free medium for bony fish leucocyte in vitro mitogern-induced proliferation", 《FISH & SHELLFISH IMMUNOLOGY》, 31 December 1991 (1991-12-31) * |
| 丰培金，张俊辉等: "鲤鱼外周血白细胞的分离和体外培养", 《中国兽医学报》, vol. 24, no. 4, 31 July 2004 (2004-07-31) * |
| 厉广坤，鄢采芹等: "无小牛血清外周血淋巴细胞培养液的研究", 《中国优生与遗传杂志》, vol. 7, no. 2, 25 April 1999 (1999-04-25) * |
| 曹丽萍，殷国俊等: "香菇多糖对鲤鱼离体培养免疫细胞的活性调节作用", 《农业生物技术学报》, vol. 16, no. 4, 30 April 2008 (2008-04-30) * |
| 逢越, 李庆伟: "日本七腮鳗外周血细胞显微结构及类淋巴细胞体外培养", 《海洋科学》, no. 1, 31 January 2012 (2012-01-31) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110438075A (en) * | 2019-08-13 | 2019-11-12 | 贵州省水产研究所 | The sturgeon leucocyte separation method of simple and efficient |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103289953B (en) | 2015-10-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sakai | Opsonization by fish antibody and complement in the immune phagocytosis by peritoneal exudate cells isolated from salmonid fishes | |
| Michel et al. | Production of viable cultures of Flavobacterium psychrophilum: approach and control | |
| US9481866B2 (en) | Methods of producing T cell populations enriched for stable regulatory T-cells | |
| CN104450616A (en) | Co-culture method of DC cell and CIK cell | |
| EP2068913A2 (en) | Immune modulators, preparations and compositions including immune modulators, tests for evaluating the activity of immune modulators and preparations and compositions including the same, and methods | |
| WO2003012058A2 (en) | Media and methods for cultivation of microorganisms | |
| JPS60256377A (en) | Utilization of phagocyte cell line | |
| CN105713873A (en) | Serum-free medium for vitro amplification culture of immune cells and application thereof | |
| CN106591231B (en) | BCG polysaccharide nucleic acid for promoting proliferation and differentiation of CIK cells, culture medium, culture method and application | |
| Cushion et al. | The persistent challenge of pneumocystis growth outside the mammalian lung: past and future approaches | |
| CN111321188A (en) | Formula for modifying antibody glycoform, cell culture method and application in industrial production | |
| JPH09176043A (en) | Vaccine and diagnostic agent for iridovirus infectious disease for fish and production of the same and the like | |
| CN103289953B (en) | Fish leukocyte separation method | |
| Moskowitz et al. | Sensitivity of cultured mammalian cells to streptomycin and dihydrostreptomycin | |
| McKinney et al. | Damselfish with neurofibromatosis exhibit cytotoxicity towards retrovirus infected cells | |
| CN105255827B (en) | One kind being used for the lymphopoietic culture medium of left-eyed flounder | |
| CN104293734A (en) | Preparation method of human gamma delta T cell | |
| RU2451743C2 (en) | Method of producing tularemic microbe biomass | |
| CN106893691B (en) | Shrimp lymphocytes, methods for purification thereof and methods for in vitro assessment of innate immune response | |
| RU2609771C1 (en) | Method for obtaining erythrocyte antigen for diagnostics of nectobacteriosis in animals | |
| RU2806156C1 (en) | Method of modeling mycobacterium tuberculosis biofilms in vitro (options) | |
| Baernstein et al. | The rate of reproduction of Entamoeba histolytica in microcultures from inocula of single trophozoites in cell-free medium prepared from embryos of the chick | |
| Lyon Jr | Growth stimulation of tissue culture cells derived from patients with neuroblastoma | |
| Vij | Using Isopycnic Equilibrium Sedimentation to Explore Cryptococcus neoformans Biology | |
| RU2193061C1 (en) | Nutrient medium for tuberculosis mycobacterium culturing (mbt-broth) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151007 Termination date: 20200524 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |