Andrade W N, Johnston M G, Hay J B
Department of Pathology, University of Toronto, Ontario, Canada.
Immunol Invest. 1996 Sep-Nov;25(5-6):455-67. doi: 10.3109/08820139609055735.
A method has been devised for labeling whole blood with the fluorescent dye fluorescein isothiocyanate (FITC) so the migration of blood lymphocytes can be studied in the sheep. Although lymphocytes can be purified from blood using density gradient media or elutriation it is difficult to obtain a large number of cells, because many cells are usually lost during the purification steps. It is desirable to label at least 10(8)-10(9) lymphocytes for lymphocyte tracking studies, because a smaller number is difficult to subsequently detect and quantitate in blood and lymph even using flow cytometry. Also, it is desirable to minimize the in vitro manipulation of lymphocytes, because dead or damaged lymphocytes will not recirculate. By labeling all the cellular components of a sample of whole blood rather than first purifying the lymphocytes we have been able to satisfy both of these criteria. Although labeled blood cells of all types are reinjected into the animal, the lymphocytes are easily distinguishable from other cells using a flow cytometer. In these studies between 2.4-12.4 x 10(8) lymphocytes were injected intravenously, and they were detectable in the blood and lymph for at least 10 days. The recovery of FITC-labeled (FITC+) lymphocytes in efferent lymph is comparable to that of lymphocytes labeled with other fluorescent or radioactive markers. The presence of labeled non-lymphoid cells in the animal makes this technique impractical for studies of lymphocyte localization within histologic sections. However, it is useful for studies in animals in which lymphatic vessels can be cannulated and the blood-to-lymph recirculation of labeled lymphocytes monitored, and it also may be applicable for studies in which lymphoid organ suspensions are analyzed using flow cytometry.
已设计出一种用荧光染料异硫氰酸荧光素（FITC）标记全血的方法，以便在绵羊中研究血液淋巴细胞的迁移。虽然可以使用密度梯度介质或淘析法从血液中纯化淋巴细胞，但很难获得大量细胞，因为在纯化步骤中通常会损失许多细胞。对于淋巴细胞追踪研究而言，标记至少10⁸ - 10⁹个淋巴细胞是理想的，因为即使使用流式细胞术，数量较少的淋巴细胞随后在血液和淋巴中也难以检测和定量。此外，尽量减少淋巴细胞的体外操作是理想的，因为死亡或受损的淋巴细胞不会再循环。通过标记全血样本的所有细胞成分，而不是先纯化淋巴细胞，我们能够满足这两个标准。尽管将所有类型的标记血细胞重新注入动物体内，但使用流式细胞仪很容易将淋巴细胞与其他细胞区分开来。在这些研究中，静脉注射了2.4 - 12.4×10⁸个淋巴细胞，并且它们在血液和淋巴中至少可检测10天。FITC标记（FITC⁺）淋巴细胞在输出淋巴中的回收率与用其他荧光或放射性标记物标记的淋巴细胞相当。动物体内存在标记的非淋巴细胞使得该技术对于组织学切片内淋巴细胞定位的研究不切实际。然而，它对于可以插管淋巴管并监测标记淋巴细胞的血液到淋巴再循环的动物研究是有用的，并且它也可能适用于使用流式细胞术分析淋巴器官悬浮液的研究。
