Pienkowski M M, Lyerly M M, Miller H C
J Immunol Methods. 1978;24(1-2):163-73. doi: 10.1016/0022-1759(78)90097-2.
A microculture assay utilizing [3H]uridine incorporation was developed to test murine spleen lymphocyte immunoreactivity in vitro. Parameters of the culture technique which included cell density, doses of LPS, Con A, PHA, [3H]uridine levels, and length of culture time were investigated. Responses were detectable at 4 h for all 3 mitogens, with labeling ranging up to 180% of the control value. By 8 h there was a 200--350% increase in mitogen-induced incorporation of radioactivity. Similar increases were observed in a serum-free system. The responses were the result of increased incorporation of label by stimulated cultures rather than decreased labeling of non-mitogen treated cultures over time. The [3H]uridine incorporation was demonstrated to be the selective response of T or B cell populations when stimulated with appropriate lectins. This assay detects early RNA synthesis, as supported by experimental observations in which accumulation of radioactivity in stimulated lymphocytes was TCA precipitable, resistant to SDS treatment, and inhibited by actinomycin D.
开发了一种利用[3H]尿苷掺入的微培养测定法，以在体外测试小鼠脾淋巴细胞的免疫反应性。研究了培养技术的参数，包括细胞密度、LPS、Con A、PHA的剂量、[3H]尿苷水平和培养时间长度。所有3种促有丝分裂原在4小时时均可检测到反应，标记范围高达对照值的180%。到8小时时，促有丝分裂原诱导的放射性掺入增加了200%-350%。在无血清系统中观察到类似的增加。这些反应是受刺激培养物中标记掺入增加的结果，而不是随着时间推移非促有丝分裂原处理培养物的标记减少。当用适当的凝集素刺激时，[3H]尿苷掺入被证明是T或B细胞群体的选择性反应。该测定法检测早期RNA合成，这得到了实验观察的支持，即在受刺激淋巴细胞中放射性积累是三氯乙酸可沉淀的，对SDS处理有抗性，并被放线菌素D抑制。
