Figdor C G, Van Es W L, Leemans J M, Bont W S
J Immunol Methods. 1984 Mar 30;68(1-2):73-87. doi: 10.1016/0022-1759(84)90138-8.
One of the major disadvantages of centrifugal elutriation (CE) is the relatively large volume (150 ml) of the various fractions, especially if small numbers of cells have to be separated and the fractions contain few cells. To reduce the volume of the fractions 2 elutriator rotors were coupled in series. Since the rotor speed of the second rotor was always kept 750 rpm higher than that of the first rotor, cells elutriated from rotor 1 were collected in rotor 2. After elutriation of a complete fraction from rotor 1, and collection in rotor 2, the cells were harvested from rotor 2. This was achieved by means of a flow distribution unit (FDU), which made it possible to disconnect the flow of both rotors and simultaneously reverse the flow of the second rotor. It is demonstrated that 40-95 X 10(6) mononuclear leukocytes may be fractionated without loss of resolution in fractions of only 9 ml. The lymphocyte (greater than 99%) and monocyte subpopulations (88-94%) obtained were as pure as with CE carried out with only 1 rotor. In addition, the cells in rotor 2 could be washed and suspended in culture medium prior to harvesting by means of the FDU. In this way loss of cells by additional centrifugation steps was avoided. Erythrocytes (RBC) present in certain lymphocyte fractions were lysed with NH4Cl and after lysis of the RBC and elution of ghosts and debris, the cells were washed and harvested. This procedure did not affect cell viability and the PHA response of the lymphocytes. The versatile system described made it possible to apply CE for the separation of small numbers of cells without loss of resolution, and demonstrated that CE is ideally suitable for concentration and washing of cells, and removal of contaminating RBC, not affecting the recovery, viability and function of the cells.
离心淘析（CE）的一个主要缺点是各个组分的体积相对较大（150毫升），特别是当需要分离少量细胞且各组分中细胞数量很少时。为了减小组分的体积，将两个淘析转子串联连接。由于第二个转子的转速总是比第一个转子高750转/分钟，从转子1淘析出来的细胞被收集到转子2中。在从转子1淘析出一个完整的组分并收集到转子2中后，从转子2中收获细胞。这是通过一个流量分配单元（FDU）实现的，该单元可以断开两个转子的流动并同时反转第二个转子的流动。结果表明，40 - 95×10⁶个单核白细胞可以在仅9毫升的组分中进行分离而不损失分辨率。所获得的淋巴细胞（大于99%）和单核细胞亚群（88 - 94%）与仅使用1个转子进行的CE一样纯净。此外，在通过FDU收获之前，转子2中的细胞可以用培养基洗涤并悬浮。通过这种方式避免了因额外的离心步骤而导致的细胞损失。某些淋巴细胞组分中存在的红细胞（RBC）用氯化铵裂解，在红细胞裂解、洗脱空泡和碎片后，对细胞进行洗涤和收获。该程序不影响细胞活力和淋巴细胞的PHA反应。所描述的通用系统使得在不损失分辨率的情况下应用CE分离少量细胞成为可能，并证明CE非常适合细胞的浓缩和洗涤以及去除污染的RBC，而不会影响细胞的回收率、活力和功能。
