CN105928753A - 流式细胞仪校准用鸡红细胞的制备方法 - Google Patents
流式细胞仪校准用鸡红细胞的制备方法 Download PDFInfo
- Publication number
- CN105928753A CN105928753A CN201610242531.0A CN201610242531A CN105928753A CN 105928753 A CN105928753 A CN 105928753A CN 201610242531 A CN201610242531 A CN 201610242531A CN 105928753 A CN105928753 A CN 105928753A
- Authority
- CN
- China
- Prior art keywords
- preparation
- flow cytometer
- red blood
- blood cell
- chicken
- 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
- 241000287828 Gallus gallus Species 0.000 title claims abstract description 52
- 210000003743 erythrocyte Anatomy 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 210000004369 blood Anatomy 0.000 claims abstract description 4
- 239000008280 blood Substances 0.000 claims abstract description 4
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 15
- 239000011734 sodium Substances 0.000 claims description 12
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 229910052708 sodium Inorganic materials 0.000 claims description 9
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 6
- 229920002307 Dextran Polymers 0.000 claims description 4
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 4
- VTAJIXDZFCRWBR-UHFFFAOYSA-N Licoricesaponin B2 Natural products C1C(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2)C(O)=O)C)(C)CC2)(C)C2C(C)(C)CC1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O VTAJIXDZFCRWBR-UHFFFAOYSA-N 0.000 claims description 4
- LPLVUJXQOOQHMX-UHFFFAOYSA-N glycyrrhetinic acid glycoside Natural products C1CC(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2=O)C(O)=O)C)(C)CC2)(C)C2C(C)(C)C1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O LPLVUJXQOOQHMX-UHFFFAOYSA-N 0.000 claims description 4
- 229960004949 glycyrrhizic acid Drugs 0.000 claims description 4
- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 claims description 4
- 239000001685 glycyrrhizic acid Substances 0.000 claims description 4
- 235000019410 glycyrrhizin Nutrition 0.000 claims description 4
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 claims description 4
- 229920000056 polyoxyethylene ether Polymers 0.000 claims description 4
- 229940051841 polyoxyethylene ether Drugs 0.000 claims description 4
- 229920000259 polyoxyethylene lauryl ether Polymers 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- -1 polyoxyethylene lauryl ether Polymers 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 2
- 229920006324 polyoxymethylene Polymers 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 11
- 206010028980 Neoplasm Diseases 0.000 abstract description 4
- 238000012356 Product development Methods 0.000 abstract description 4
- 230000008859 change Effects 0.000 abstract description 3
- 239000003795 chemical substances by application Substances 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000005406 washing Methods 0.000 abstract description 2
- 238000005119 centrifugation Methods 0.000 abstract 1
- 239000003761 preservation solution Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 12
- 210000000170 cell membrane Anatomy 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- 239000004141 Sodium laurylsulphate Substances 0.000 description 3
- 230000009514 concussion Effects 0.000 description 3
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000012666 negative regulation of transcription by glucose Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000013097 stability assessment Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/1012—Calibrating particle analysers; References therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N2001/2893—Preparing calibration standards
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/1012—Calibrating particle analysers; References therefor
- G01N2015/1016—Particle flow simulating, e.g. liquid crystal cell
Landscapes
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Dispersion Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本案涉及流式细胞仪校准用鸡红细胞的制备方法,包括:将采集到的鸡静脉血加入到等体积的分离剂中混合3‑6min;200‑500rpm离心,去上清,得到鸡红细胞;采用磷酸缓冲液洗涤所获得的鸡红细胞;将鸡红细胞分散于保存液中。本案中的鸡红细胞制备方法仅需两步即可完成,可作为很好的产品开发方案;将逐步改变国内流式细胞仪用试剂对进口试剂的依赖,大大降低流式细胞仪的应用成本;与此同时,该试剂的大量生产可推动临床肿瘤倍体检测的方便进行,提高肿瘤治疗质量;所获得的鸡红细胞产品可在常温下可至少保存3个月,4℃下则可保存至少1年以上,且所有试剂均可在带菌条件下进行,从而避免了无菌操作的复杂步骤,降低细胞制备成本。
Description
技术领域
本发明属于医疗检测用试剂领域,具体涉及一种流式细胞仪校准用鸡红细胞的制备方法。
背景技术
流式细胞仪是目前生命科学领域最先进的仪器之一,可高通量的提供细胞多级信息。为保证准确性,流式细胞仪在使用前均需使用标准试剂进行校准,但在实际使用中,流式细胞仪需要采用校准试剂来进行校准与稳定性分析,之后才能进行样品检测。鸡红细胞是早期流式细胞仪校准与稳定性评估的主要试剂之一,即便在更稳定的微球质控品出现后,其仍可被用于流式细胞仪的粗略校准,从而节省微球的用量,降低仪器应用成本。同时,由于鸡红DNA含量稳定为正常人体细胞的1/3,可在流式细胞仪上与人正常细胞或超二倍体细胞峰完全分开,因而可被用于检测肿瘤的诊断及判定预后。然而目前市售的流式细胞仪用鸡红细胞主要来至于进口,价格昂贵且供货周期长。给流式细胞仪的应用来了很多不便。
目前国内外尚无公开的鸡红细胞产品开发方案,有限的文献报道中所提供的制备方法往往倾向于仅采用磷酸缓冲盐溶液洗涤及离心的方法进行,但采用这种手段获得的产品在流式细胞仪上的分辨率较低,仅能用于粗略校准,且受限于不同的实验环境,各研究组的检测结果也有所差别,无法作为产品开发的标准依据。
发明内容
针对现有技术存在的不足,本发明的目的在于提供了一种流式细胞仪校准用鸡红细胞的制备方法,其能够作为一种标准方法,方便而稳定地制备鸡红细胞,从而配合流式细胞仪使用,并可作为产品开发的标准依据。
为实现上述目的,本发明通过以下技术方案实现:
一种流式细胞仪校准用鸡红细胞的制备方法,其包括:
将采集到的鸡静脉血加入到等体积的分离剂中混合3-6min;
200-500rpm离心,去上清,得到鸡红细胞;
采用磷酸缓冲液洗涤所获得的鸡红细胞;
将鸡红细胞分散于保存液中。
优选的是,所述的流式细胞仪校准用鸡红细胞的制备方法,其中,所述分离剂包括有0.01-0.05M的Na2HPO4、0.01-0.05M的NaH2PO4、0.5-2wt%的葡萄糖、6-12wt%的右旋糖酐、0.1-0.3wt%的乙二胺四乙酸二钠、0.1-1wt%的甘氨酸、1-3wt%的甘草酸和0.1-0.3wt%的月桂醇聚氧乙烯醚。
优选的是,所述的流式细胞仪校准用鸡红细胞的制备方法,其中,所述保存液包括有1-2wt%的甘油、0.1-0.3wt%的叠氮钠、3-5wt%的多聚甲醛、0.1-0.3wt%的曲拉通和0.01-0.03wt%的聚氧乙烯醚。
优选的是,所述的流式细胞仪校准用鸡红细胞的制备方法,其中,所述分离剂的pH为7.3-7.5。
优选的是,所述的流式细胞仪校准用鸡红细胞的制备方法,其中,所述保存液还包括有0.01-0.03wt%的脱氢乙酸钠。
本发明的有益效果是:
1、本案中的鸡红细胞制备方法仅需两步即可完成,同时所获得的产品可长期保存,因此可很好的作为产品开发方案。
2、逐步改变国内流式细胞仪用试剂对进口试剂的依赖,大大降低流式细胞仪的应用成本。与此同时,该试剂的大量生产可推动临床肿瘤倍体检测的方便进行,提高肿瘤治疗质量。
3、本方法中所获得的鸡红细胞产品可在常温下可至少保存3个月,4℃下则可保存至少1年以上,且所有试剂均可在带菌条件下进行,从而避免了无菌操作的复杂步骤,降低细胞制备成本。
附图说明
图1是采用流式细胞仪校准用鸡红细胞的制备方法所获鸡红细胞的400倍显微镜图。
图2是采用流式细胞仪校准用鸡红细胞的制备方法所获鸡红细胞的200倍显微镜图。
图3是采用流式细胞仪校准用鸡红细胞的制备方法所获的鸡红细胞在常温下保存90天后的流式细胞仪检测结果图;图3表明鸡红细胞即便在保存了90天后,依然具备很好的校准性能。
具体实施方式
下面结合附图对本发明做进一步的详细说明,以令本领域技术人员参照说明书文字能够据以实施。
本案列出一实施例的流式细胞仪校准用鸡红细胞的制备方法,其包括:
将采集到的鸡静脉血加入到等体积的分离剂中混合3-6min;
200-500rpm离心,去上清,得到鸡红细胞;
采用磷酸缓冲液洗涤所获得的鸡红细胞;
将鸡红细胞分散于保存液中。
其中,分离剂包括有0.01-0.05M的Na2HPO4、0.01-0.05M的NaH2PO4、0.5-2wt%的葡萄糖、6-12wt%的右旋糖酐、0.1-0.3wt%的乙二胺四乙酸二钠、0.1-1%的甘氨酸、1-3wt%的甘草酸和0.1-0.3wt%的月桂醇聚氧乙烯醚,其余为水。Na2HPO4和NaH2PO4起到缓冲液的作用,使pH值维持在7.3-7.5之间;葡萄糖在这里的作用是能量试剂,用于给细胞供给能量;右旋糖酐在这里的作用是为了让红细胞沉降,与白细胞分离;乙二胺四乙酸二钠是一种细胞分散试剂;甘氨酸是细胞膜表面蛋白的稳定剂;甘草酸在这里是一种抗氧化剂,其主要用于治疗细胞在分离过程中易发生的过氧化损伤;月桂醇聚氧乙烯醚作为一种抗震保护剂,主要作用在于降低分离过程中存在的切削力损伤。
在上述实施例中,保存液包括有1-2wt%的甘油、0.1-0.3wt%的叠氮钠、3-5wt%的多聚甲醛、0.1-0.3wt%的曲拉通和0.01-0.03wt%聚氧乙烯醚,其余为水。甘油的作用有两点:1)细胞膜保护剂,甘油可以增加溶液粘度,从而降低对细胞的震荡,同时,它降低体系极性,保持细胞膜的流动性和膜上蛋白的稳定性;2)防震剂,其主要用于在运输过程中减小因震荡造成的细胞损坏。叠氮钠在这里是一种抗菌剂与防腐剂;多聚甲醛是一种固定剂,其通过使细胞膜表面蛋白变性来稳定结构,使细胞的构型不会再发生变化。曲拉通的作用是:1)细胞膜通透剂,其可在细胞膜上制造小孔,方便染色剂进入;2)防震剂,其主要用于在运输过程中减小因震荡造成的细胞损坏。聚氧乙烯醚作用则为稳定细胞骨架及缓解固定剂对细胞的损伤,同时具有稳定染色效果,使流式细胞仪检测结果保持在稳定范围内。
在上述实施例中,分离剂的pH优选为7.3-7.5。
作为本案另一实施例,其中,保存液还优选包括有0.01-0.03wt%的脱氢乙酸钠。脱氢乙酸钠是一种防变质剂,其用于在运输过程中,当冷藏系统发生意外故障时,储藏温度会升高,脱氢乙酸钠可以用于短期内保证细胞不发生变质,但其所能承受的防变质温度上限为40℃,若不添加脱氢乙酸钠,正常的鸡红细胞在40℃下仅能保持5分钟的有效性,而当添加脱氢乙酸钠后,其维持时间可以达到半小时,以此来争取到足够的时间对冷藏系统进行修复或对鸡红细胞进行应急转移处理。但脱氢乙酸钠的用量应被限制,若超出优选的范围,将会抑制曲拉通对细胞膜的通透作用。
将得到的鸡红细胞进行碘化丙啶染色,经流式细胞仪检测,CV值在6%以下的,可用于后续试验。
对比试验:
(1)在上述实施例(未添加脱氢乙酸钠)中,将曲拉通替换为同比例的SDS(十二烷基硫酸钠)或吐温:
保存时间 | 常温 | 4℃ |
曲拉通 | 90d | 370d |
SDS | 75d | 300d |
吐温 | 80d | 320d |
(2)在上述实施例(未添加脱氢乙酸钠)中,将多聚甲醛替换为同比例的戊二醛、甲醛或70%冰乙醇:
尽管本发明的实施方案已公开如上,但其并不仅仅限于说明书和实施方式中所列运用,它完全可以被适用于各种适合本发明的领域,对于熟悉本领域的人员而言,可容易地实现另外的修改,因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节和这里示出与描述的图例。
Claims (5)
1.一种流式细胞仪校准用鸡红细胞的制备方法,其特征在于,包括:
将采集到的鸡静脉血加入到等体积的分离剂中混合3-6min;
200-500rpm离心,去上清,得到鸡红细胞;
采用磷酸缓冲液洗涤所获得的鸡红细胞;
将鸡红细胞分散于保存液中。
2.如权利要求1所述的流式细胞仪校准用鸡红细胞的制备方法,其特征在于,所述分离剂包括有0.01-0.05M的Na2HPO4、0.01-0.05M的NaH2PO4、0.5-2wt%的葡萄糖、6-12wt%的右旋糖酐、0.1-0.3wt%的乙二胺四乙酸二钠、0.1-1wt%的甘氨酸、1-3wt%的甘草酸和0.1-0.3wt%的月桂醇聚氧乙烯醚。
3.如权利要求1所述的流式细胞仪校准用鸡红细胞的制备方法,其特征在于,所述保存液包括有1-2wt%的甘油、0.1-0.3wt%的叠氮钠、3-5wt%的多聚甲醛、0.1-0.3wt%的曲拉通和0.01-0.03wt%的聚氧乙烯醚。
4.如权利要求1所述的流式细胞仪校准用鸡红细胞的制备方法,其特征在于,所述分离剂的pH为7.3-7.5。
5.如权利要求3所述的流式细胞仪校准用鸡红细胞的制备方法,其特征在于,所述保存液还包括有0.01-0.03wt%的脱氢乙酸钠。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610242531.0A CN105928753B (zh) | 2016-04-18 | 2016-04-18 | 流式细胞仪校准用鸡红细胞的制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610242531.0A CN105928753B (zh) | 2016-04-18 | 2016-04-18 | 流式细胞仪校准用鸡红细胞的制备方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN105928753A true CN105928753A (zh) | 2016-09-07 |
CN105928753B CN105928753B (zh) | 2019-01-15 |
Family
ID=56838433
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610242531.0A Active CN105928753B (zh) | 2016-04-18 | 2016-04-18 | 流式细胞仪校准用鸡红细胞的制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105928753B (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108548771A (zh) * | 2018-06-11 | 2018-09-18 | 天晴干细胞股份有限公司 | 一种用于流式检测的细胞固定液及其使用方法 |
CN113432959A (zh) * | 2021-05-21 | 2021-09-24 | 赛雷纳(中国)医疗科技有限公司 | 一种精子dna碎片化检测用质控品的制备方法 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4704891A (en) * | 1986-08-29 | 1987-11-10 | Becton, Dickinson And Company | Method and materials for calibrating flow cytometers and other analysis instruments |
CN1501084A (zh) * | 2002-11-14 | 2004-06-02 | 北京市红十字血液中心 | 试剂红细胞 |
CN101289493A (zh) * | 2008-04-22 | 2008-10-22 | 中国人民解放军第三军医大学野战外科研究所 | 一种从猪血中提取高纯度血红蛋白的方法 |
CN103540565A (zh) * | 2012-07-17 | 2014-01-29 | 苏州生物医学工程技术研究所 | 一种鸡红细胞的制备和储存方法 |
CN105324479A (zh) * | 2013-04-15 | 2016-02-10 | Cells4Life集团公司 | 细胞分离的方法 |
-
2016
- 2016-04-18 CN CN201610242531.0A patent/CN105928753B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4704891A (en) * | 1986-08-29 | 1987-11-10 | Becton, Dickinson And Company | Method and materials for calibrating flow cytometers and other analysis instruments |
CN1501084A (zh) * | 2002-11-14 | 2004-06-02 | 北京市红十字血液中心 | 试剂红细胞 |
CN101289493A (zh) * | 2008-04-22 | 2008-10-22 | 中国人民解放军第三军医大学野战外科研究所 | 一种从猪血中提取高纯度血红蛋白的方法 |
CN103540565A (zh) * | 2012-07-17 | 2014-01-29 | 苏州生物医学工程技术研究所 | 一种鸡红细胞的制备和储存方法 |
CN105324479A (zh) * | 2013-04-15 | 2016-02-10 | Cells4Life集团公司 | 细胞分离的方法 |
Non-Patent Citations (2)
Title |
---|
殷建等: "流式细胞仪用鸡红细胞的制备", 《J SOUTH MED UNIV》 * |
胡凡等: "大型流式细胞仪质量控制方法的探讨", 《南京医科大学学报》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108548771A (zh) * | 2018-06-11 | 2018-09-18 | 天晴干细胞股份有限公司 | 一种用于流式检测的细胞固定液及其使用方法 |
CN113432959A (zh) * | 2021-05-21 | 2021-09-24 | 赛雷纳(中国)医疗科技有限公司 | 一种精子dna碎片化检测用质控品的制备方法 |
Also Published As
Publication number | Publication date |
---|---|
CN105928753B (zh) | 2019-01-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Yang et al. | Malondialdehyde level and some enzymatic activities in subclinical mastitis milk | |
Beutler et al. | Biochemical and electrophoretic studies of-galactosidase in normal man, in patients with Fabry's disease, and in Equidae. | |
Meyer | Mucoids and glycoproteins | |
Kalaitzakis et al. | Clinicopathological evaluation of downer dairy cows with fatty liver | |
WO2018001215A1 (zh) | 检测细菌性阴道病的试剂盒 | |
Michell et al. | The comparative effectiveness of three commercial oral solutions in correcting fluid, electrolyte and acid-base disturbances caused by calf diarrhoea | |
EP4116401A1 (en) | Storage container for cell-containing solution and storage solution | |
CN105928753A (zh) | 流式细胞仪校准用鸡红细胞的制备方法 | |
Bogin et al. | Distribution of lactate dehydrogenase isoenzymes in normal and inflamed bovine udders and milk | |
CN104585162B (zh) | 一种新型环保无毒的液基细胞保存液 | |
Gilbert et al. | Impaired post partum neutrophil function in cows which retain fetal membranes | |
Pandiar et al. | Use of jaggery and honey as adjunctive cytological fixatives to ethanol for oral smears | |
Alam et al. | Effects of Chitosan-oligosaccharide on diarrhoea in Hanwoo calves. | |
CN105432598A (zh) | 一种液基细胞保存液及其制备方法 | |
Kandemir et al. | A different approach to diagnosis of subclinical mastitis: milk arginase activity. | |
CN101718783B (zh) | 广谱抗人球蛋白卡的制备方法 | |
Hall et al. | The use of dextran sulphate as a blood anticoagulant in biological research | |
Ozturk et al. | Local oxidative stress in interdigital tinea pedis | |
CN113373031B (zh) | 一种喷雾型的游离dna样本保存管及应用 | |
CN115777695A (zh) | 适用于脑脊液细胞形态学检查的复合保存液及其制备方法 | |
CN109321561B (zh) | 一种用于保护体外血液中核酸的保存剂及采血管 | |
Pinto-Pinho et al. | Mitochondrial Effects, DNA Damage, and Antioxidant Enzyme Activity in Cryopreserved Human Sperm Samples: A Pilot Study | |
Kitchen et al. | Enzymic methods for the estimation of the somatic cell count in bovine milk: II. N-acetyl-β-D-glucosaminidase test for routine estimation of the somatic cell count in milk | |
JP5330543B2 (ja) | 正常新鮮血小板の模擬物 | |
CN107583699A (zh) | 一种用于医学临床检验的采血管及其制备方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |