Varesio L, Naglich J, Brunda M J, Taramelli D, Eva A
Immunol Commun. 1981;10(7):577-89. doi: 10.3109/08820138109050711.
A method is described for the evaluation of the total 3H-uridine incorporated by macrophages in vitro into trichloroacetic acid (TCA)-precipitable material. The technique is based upon solubilization of the macrophage monolayers by guanidine-HCl, followed by TCA precipitation. The recovery of RNA into the precipitate and the reproducibility of the results were strictly dependent on the use of filtered reagents and on incubation of the TCA precipitate for 2 or more hours at 4 degree C before harvesting. Treatment with quanidine-HCl did not affect the recovery of labeled RNA. Moreover, we observed that radioactive precipitate had the characteristics of RNA, since its recovery was sensitive to the addition of unlabeled uridine in the culture medium and to the treatment of the macrophages with inhibitors of RNA synthesis, but not of protein synthesis. Tritiated uridine incorporation in microcultures of macrophages can be assessed with this technique, by processing the cells directly in the wells. The main advantages of this procedure are: 1) the radioactivity can be measured by semiautomatic cell harvesters, 2) a small number of macrophages are required, and 3) many samples can be processed simultaneously. Overall, the technique is simple, rapid, and could be successfully adapted to study other metabolic pathways.
本文描述了一种评估巨噬细胞在体外将总3H-尿苷掺入三氯乙酸（TCA）沉淀物质中的方法。该技术基于用盐酸胍溶解巨噬细胞单层，然后进行TCA沉淀。RNA在沉淀物中的回收率以及结果的可重复性严格取决于使用过滤后的试剂，以及在收获前将TCA沉淀物在4℃下孵育2小时或更长时间。用盐酸胍处理不会影响标记RNA的回收率。此外，我们观察到放射性沉淀物具有RNA的特征，因为其回收率对培养基中未标记尿苷的添加以及用RNA合成抑制剂而非蛋白质合成抑制剂处理巨噬细胞敏感。通过直接在孔中处理细胞，可用该技术评估巨噬细胞微培养物中氚标记尿苷的掺入情况。该方法的主要优点是：1）放射性可通过半自动细胞收获仪测量；2）所需巨噬细胞数量少；3）可同时处理多个样品。总体而言，该技术简单、快速，并且可以成功地用于研究其他代谢途径。
