Lattime E C, Stoppacciaro A, Stutman O
Immunology Program, Memorial Sloan-Kettering Cancer Center, New York 10021.
J Immunol. 1988 Nov 15;141(10):3422-8.
A limiting dilution assay (LDA) that measures the frequency of TNF producing cells is described. LDA determination is based on the inhibition of growth of a highly TNF sensitive subline from the WEHI-164 fibrosarcoma by using a micro assay sensitive to single picogram amounts of recombinant murine TNF. Using such LDA, it was determined that the reported deficiency in LPS-induced TNF production in C3H/HeJ mice is a function of reduced frequency of TNF producing cells rather than a complete lack of responsiveness. In bulk culture, LPS-triggered TNF was produced by Thy-1.2 negative spleen cells with activity recovered in both G10 Sephadex adherent and nonadherent subpopulations. LPS stimulation of spleen cells from C3H/HeJ mice resulted in TNF mRNA expression as shown in both Northern blots and in situ hybridization. The frequency of TNF mRNA bearing cells in control of C3H/HeSnJ mice by in situ hybridization correlated with that found for TNF producing cells in LDA. In C3H/HeJ spleen, significantly higher numbers of TNF mRNA positive cells were found than were shown to produce TNF in LDA.
本文描述了一种用于测量产生肿瘤坏死因子（TNF）细胞频率的有限稀释分析（LDA）方法。LDA测定基于使用对单皮克量重组小鼠TNF敏感的微量分析，通过抑制来自WEHI-164纤维肉瘤的高度TNF敏感亚系的生长来进行。使用这种LDA方法，确定了报道的C3H/HeJ小鼠中脂多糖（LPS）诱导的TNF产生缺陷是由于产生TNF细胞频率降低，而非完全缺乏反应性。在批量培养中，LPS触发的TNF由Thy-1.2阴性脾细胞产生，其活性在G10葡聚糖凝胶粘附和非粘附亚群中均有恢复。LPS刺激C3H/HeJ小鼠的脾细胞导致TNF mRNA表达，这在Northern印迹和原位杂交中均有显示。通过原位杂交检测，C3H/HeSnJ小鼠对照组中携带TNF mRNA的细胞频率与LDA中产生TNF的细胞频率相关。在C3H/HeJ脾中，发现TNF mRNA阳性细胞的数量明显高于LDA中显示产生TNF的细胞数量。
