Strauss P R, Sheehan J M, Kashket E R
J Exp Med. 1976 Oct 1;144(4):1009-21. doi: 10.1084/jem.144.4.1009.
We have developed a rapid sampling technique for animal cells in suspension for the purpose of measuring membrane transport in lymphocytes. The method involves rapid centrifugation of cells through a layer of silicone oil into perchloric acid after incubation periods as short as 4 s. Using this method we have described the uptake of thymidine and the uptake and transport systems of adenosine by murine bulk nonadherent spleen cells. The two uptake systems are markedly different. Adenosine was shown to be taken up by classical carrier-mediated diffusion, while thymidine was not. In addition we have explored the metabolism of the two nucleosides under the conditions we employed for measuring transport or uptake; Both nucleosides are phosphorylated extensively. We also investigated the uptake and metabolism of thymidine over a 2-h interval the standard time used to measure DNA synthesis in lymphocytes. Unless cells were separated from medium by centrifugation through oil before TCA addition, the TCA precipitable counts exceeded the total radioactive uptake. Hence the standard method for measuring thymidine utilization yields estimates under these conditions which can be as much as 100% too high.
为了测量淋巴细胞中的膜转运，我们开发了一种针对悬浮动物细胞的快速采样技术。该方法包括在短至4秒的孵育期后，通过一层硅油将细胞快速离心到高氯酸中。使用这种方法，我们描述了小鼠大量非贴壁脾细胞对胸苷的摄取以及腺苷的摄取和转运系统。这两种摄取系统明显不同。结果表明，腺苷通过经典的载体介导扩散被摄取，而胸苷则不是。此外，我们还研究了在用于测量转运或摄取的条件下这两种核苷的代谢情况；两种核苷都被广泛磷酸化。我们还在用于测量淋巴细胞DNA合成的标准2小时时间间隔内研究了胸苷的摄取和代谢。除非在加入三氯乙酸（TCA）之前通过油层离心将细胞与培养基分离，否则TCA可沉淀计数会超过总放射性摄取量。因此，在这些条件下，测量胸苷利用率的标准方法所得到的估计值可能会高出多达100%。
