淋巴细胞分离液(LSM)LSM-Lymphocyte Separation Medium

• 产品编号：0850494X      品牌：MP       原厂货号：0850494X
• 产品分类：生化和细胞分析 > 细胞及细胞器分离 > 细胞分离液
• 应用分类：暂定分类 > 其他解离产品
  　　　　　细胞培养产品与服务 > 特定细胞培养 > 细胞分离与扩增 > 细胞分离 > 人细胞分离

描述：

LSM Lymphocyte Separation Medium
 

LSM® -6.2克聚蔗糖Ficoll和9.4克泛影酸钠/100ml；

密度 20℃ 时1.0770-1.0800克/毫升，无菌溶液；

人红细胞密度为1.093，粒细胞为1.092，淋巴细胞为1.074±0.001。密度梯度离心法。

优点：

第二代产品，泛影酸钠代替甲泛影酸钠

回收率>90%，纯度>97% ，活力>98.8%

适用收集后6小时内血液，肝素/EDTA抗凝

4–8°C 避光2年 仅适用肝素抗凝, 血液处理时间：收集后6小时内

LSM^™ is a sterile-filtered solution of a sucrose polymer and diatrizoate salts at a specific gravity of 1.077-1.080 g/mL at 20°C. LSM^™ is formulated for isolation of mononuclear cells from defribinated or heparinized whole human blood. One-step centrifugation permits separation of mononuclear lymphocytes from erythrocytes, polynuclear lymphocytes and most platelets.
Ref.: Ojaniemi, H., et al., Biotechniques, 35: 5 (2003).

Intended Use

For in vitro isolation of lymphocytes from peripheral blood.

Summary of the method

Early methods for isolating leukocytes involved mixing blood with a compound which aggregated the erythrocytes but only slightly affected leukocytes. With centrifugation, erythrocytes pelleted due to their increase density , and leukocytes were collected from the upper part of the tube.

Boyum³ introduced a more convenient and rapid separation using centrifugation through a Ficoll-sodium metrizoate solution. Diluted blood was layered over the Ficoll-sodium metrizoate solution and centrifuged at a low speed for a short time. Erythrocytes and granulocytes sedimented to the bottom of the tube, and mononuclear cells (lymphocytes) and platelets were collected from the interface between the two phases.

Modifications of the Boyum formulation have been made by numerous workers. "LSM" produced by MP Biomedicals, has a unique formulation using the successful substitution of sodium diatrizoate for the sodium metrizoate.

Principle of the procedure

Defibrinated or heparinized human blood is diluted with physiological saline or balanced salt solution in 1:1 proportion, layered over the separation medium, and centrifuged at a low speed for 30 minutes. During centrifugation, differential migration results in the formation of several cell layers.

The pellet which is formed is comprised mostly of erythrocytes and granulocytes which have migrated through the gradient. Due to their density, lymphocytes and other mononuclear cells (platelets and monocytes) are found at the plasma-LSM interface. Lymphocytes are recovered by aspirating the layer. Further washing removes the platelets, LSM, and plasma.

Reagents

LSM is a sterile filtered solution which contains 6.2g Ficoll and 9.4 g sodium diatrizoate per 100 ml. The density is 1.07700.1-1.0800 g/ml at 20^oC.

Precautions

The material is intended for LABORATORY USE ONLY for the in vitro separation of lymphocytes from peripheral blood. LSM IS NOT INTENDED FOR IN VIVO USE.

DO NOT USE IF THE MATERIAL IS CLOUDY, HAS A DISTINCT YELLOW COLOR, OR SHOWS ANY SIGNS OF CONTAMINATION.

Stability/storage

Stable until the expiration date listed on the vial. Store at room temperature (18-25^oC) in the origin carton. Protect from light.

Instructions for use

The following procedure is one of many variants of the procedure originally described by Boyum. This procedure was developed for use with defibrination or anti-coagulant treated human blood; alterations may be necessary for use with blood from other species or with other tissues.^13-22

用途：用于体外分离外周血淋巴细胞，也可以从其他来源中分离单核细胞，包括脐带血细胞和骨髓细胞。

方法概述：

早期分离白细胞的方法，会用一种化合物混合血液。此化合物能够聚集红细胞，只轻微影响白细胞。通过离心，红细胞由于密度增加而聚集沉淀，同时从离心管内上层收集白细胞。

Böyum提出了一种更方便快速的分离方法，即以Ficoll^®-甲泛影钠溶液为介质进行离心。稀释的血液在Ficoll-甲泛影钠溶液中分层，之后低速短时离心。红细胞和多形核粒细胞沉降到管底，单个核细胞（淋巴细胞）和血小板可以从两个分层间的分界面处收集。

    许多研究者不断改进了Böyum方法，对于MP生物医学公司生产的LSM，其方法的独特之处在于成功运用泛影酸钠代替了甲泛影钠。

程序原理：

去纤维蛋白或肝素化人血以1：1的比例用生理盐水或平衡盐溶液稀释，在分离溶液中分层，低速离心30min。离心过程中，由于差速迁移最终形成不同的细胞层。

    聚集在管底的颗粒主要是红细胞和多形核粒细胞，它们通过梯度迁移至管底。由于其密度不同，淋巴细胞和其他单个核细胞（单核细胞和血小板）分布在血浆和LSM两层之间。淋巴细胞可以通过吸取分界层溶液回收，最后进一步清洗去除血小板，LSM和血浆。

试剂：

LSM^® is a sterile filtered solution which contains 6.2g Ficoll and 9.4 g sodium diatrizoate per 100 ml. The density is 1.0770-1.0800 g/ml at 200C.

       LSM^® 是一种灭菌过滤溶液，每100毫升包含6.2克聚蔗糖和9.4克钠胺。密度是1.0770-1.0800克/毫升20℃。

0850494X       淋巴细胞分离液(LSM（LYMPHOCYTE SEPARATION MEDIUM ）

使用说明：

以下的操作步骤是最初由Böyum.设计的程序中众多改良版本的一种。此程序经改良用于去纤维蛋白或抗凝血剂处理过的人体血液；对于其他物种或者其他组织的血液，改变此程序也许是有必要的。

1. 轻轻颠倒瓶子使LSM充分混合

2. 无菌转移3 ml LSM到15 ml离心管中。

3. 混合2 ml去纤维蛋白血液或肝素化血液和2 ml 生理盐水

4. 仔细的将稀释血液加入含有3 ml LSM（室温）的15ml离心管中，在血液和LSM中形成一个明显的分层。不要将稀释血液混合入LSM中。

5. 室温下400xg离心15-30min，离心可以沉淀红细胞和多形核白细胞，同时可以在LSM上形成一层单核淋巴细胞，如上图所示（具体分层情况为血浆层-单个核细胞层-LSM层-RBC颗粒）。

6. 吸出淋巴细胞上方2-3mm的血浆。

7. 吸取淋巴细胞层以及它下面一半的LSM，并转移到另外一个离心管。加入等体积的平衡盐缓冲液至离心管内的淋巴细胞层中，室温（18-25°C）离心10分钟，离心速度设定在既不损伤细胞又能沉淀细胞即刻，例如160 – 260 xg（清洗去除LSM，并降低血小板的百分比）。

8. 再用平衡盐缓冲液清洗细胞，最后用适当的培养基重悬细胞。

1. Thoroughly mix the LSM by inverting the bottle gently.
2. Aseptically transfer 3 ml of LSM to a 15 ml centrifuge tube.
3. Mix 2 ml of defibrinated, heparinized blood with 2 ml of physiological saline.
4. Carefully layer the diluted blood over 3 ml of LSM (room temperature) in a 15 ml centrifuge tube, creating a sharp blood-LSM interface. DO NOT MIX DILUTED BLOOD INTO THE LSM.
5. Centrifuge the tube at 400 x g at room temperature for 15-30 minutes. Centrifugation should sediment erythrocytes and polynuclear leukocytes and band mononuclear lymphocytes above LSM (Bands will be Plasma layer -----> Mononuclear cell layer -----> LSM layer -----> RBC pellet) as shown in the diagram above.
6. Aspirate the top layer of clear plasma to within 2-3 mm above the lymphocyte layer.
7. Aspirate the lymphocyte layer plus about half of the LSM layer below it and transfer it to a centrifuge tube. Add an equal volume of buffered balanced salt solution to the lymphocyte layer in the centrifuge tube and centrifuge for 10 minutes at room temperature (18-25^oC) at a speed sufficient to sediment the cells without damage. i.e. 160-260 x g . (Washing removes LSM and reduces the percentage of platelets).
8. Wash the cells again with buffered balanced salt solution and resuspend in appropriate medium for your applications.

Store at room temperature (18-25^oC) in the original carton. Stable until the expiration date listed on the vial. Protect from light.

Caution : The material is intended for laboratory use only for the in vitro separation of lymphocytes from peripheral blood. LSM is not intended for in vivo use.

Do not use if the material is cloudy, has a distinct yellow color or shows any signs of contamination.

References
 

1. Skoog, W.A and Beck, W.S " Studies in the Fibrinogen , dextran , and phytohemagglutinin methods of isolating leukocytes." Blood 11: 436-454, 1956
2. Boyum,A. " Seperation of white blood cells." Nature 204: 793-794, 1964
3. Boyum,A " Isolation of mononuclear cells and granulocytes from human blood ." Scand.J.Clin.Lab Invest. 21, Suppl. 97 : 77, 1968
4. DeVries,J.E. can Benthem,M. and Rumke,P. " Separation of viable from non-viable tumor cells by flotation on a Ficoll-Triosil mixture." Transplantation 15 : 409, 1973
5. Harris,R. and Ukaejiofo, E.V., " Rapid preparation of lymphocytes for tissue typing ". Lancet 2. 327, 1969.
6. Kay, H.D., " A new procedure to overlay diluted blood on Ficoll-Hypaque gradients." J. Immunol. Meth. 39, 81, 1980.
7. Thorsbey, E. and BratlieA., " A rapid method for preparation of pure lymphocyte suspensions". In Histocompatibility Testing, P.I. Terasaki, ed. Munksgaard, Copenhagen, pp 664-665, 1970.
8. Ting, A. and Morris, P.J., " A technique for lymphocyte preparation from stored heparinized blood". Vox Sang, 20, 561, 1971.
9. Fotino, M., Merson, E.J. and Allen, F.H., " Micromethod for rapid separation of lymphocytes from peripheral blood". Ann.Clin. Lab. Sci.1, 131-133, 1971.
10. Fotino, M., Merson, E.J. and Allen, F.H. " Instant lymphocytes". Vox Sang 21, 469-470, 1971.
11. Wybran, J. Chanther, S. and Fudenberg, H., " Response to phytohemagglutinin". J. Immunol. 110. 1157-1160, 1973.
12. Bignold, L.P. and Ferrante, A., " Mechanism of separation of polymorphonuclear leukocytes from whole blood by the one-step Hypaque-Ficoll method" J. Immunol. Meth. 96, 29-33, 1987.
13. Boumsell, L . and Meltzer, M.S., " Mouse mononuclear cell chemotaxis : I. Differntial response of monocytes and macrophages". J. Immunol. 115, 1746, 1975.
14. Colvin, R.B. and Dvorak, H.F., " Fibrinogen/fibrin on the surface of macrophages : detection distribution, binding requirements and possible role in macrophage of adherence phenomena" J. Experimental Medicine 142, 1377, 1975.
15. Dean, J.H., Silva, J.S.m McCoy, J.L., Leonard, C.M., Cannon, G.B. and Herberman, R.B., " Functional activities of rosette separated human peripheral blood leukocytes". J. Immunol. 115, 1449, 1975.
16. Kay, H.D. and Horwitz , D.A. " Natural and antibody dependent cytotoxicity of lymohocytes and monocytes". In Methods of Immunodiagnosis, 2nd Edition ed. N.Rose and P. Bigazzi, John Wiley & Sons, Inc., New York, pp. 15-25, 1980.
17. Lett-Brown, M.A., Boetcher, D.A., and Leonard, E.J., " Chemotactic responses of normal human basophils to C5a and to lymphocyte-derived chemotactic factor." J. Immunol. 117, 246, 1976.
18. Nelson, R.D., Quie, P.G. and Simmons, R.L., " Chemotaxis under agarose : a new and simple method of measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes". J.Immunol. 115, 1650, 1975.
19. Neubaur, R.H., Wallen, W.C. and Rabin, H., " Inhibition of mitogenic response of normal peripheral lymphocytes by extracts or supernatant fluids of a herpesvirus saimiri lymphoid tumor cell line". Infection & Immunity 12(5), 1021, 1975.
20. Pollack, V.A., Brandhurst, J.S. and Hanna, Jr., M.G., " Separation of guinea pig peripheral blood lymphocytes by discontinuous density, gradient centrifugation using Ficoll-Metrizoate." J. Immunol. Meth. 41, 29, 1981.
21. Torten, M., Johnson, R.C., Kaattari, S., Leung, C. and Benjamini, E., " Immune plasma-dependent cytotoxicity of immune and non-immune peripheral lymphoid cells for target cells coated with bacterial outer unit membrane". Immunology 29, 1093, 1975.
22. Ziegler, H.K. and Henney, C.S., " Antibody dependent cytolytically active human leukoctyes : an analysis of inactivation following in vitro interaction with antibody-coated target cells". J. Immuno. 115, 1500, 1975.

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