Limiting dilution analysis of TNF producing cells in C3H/HeJ mice.
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.
D000818:Animals; D000954:Antigens, Surface; D002460:Cell Line; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods; D005260:Female; D005354:Fibrosarcoma / Q000378:metabolism; D007958:Leukocyte Count / Q000187:drug effects; D008070:Lipopolysaccharides; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D013154:Spleen / Q000166:cytology / Q000378:metabolism*; D018800:Thy-1 Antigens; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000502:physiology
D000954:Antigens, Surface; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D018800:Thy-1 Antigens; D014409:Tumor Necrosis Factor-alpha