Detection of colony-stimulating factor messenger RNA in single T cells by in situ hybridization.
In situ hybridization has been used to study the accumulation of colony-stimulating factor (CSF) mRNA in single cells of a T lymphocyte clone (E9.D4) following antibody-mediated (F23.1) activation via the Ti-T3 complex in filler-independent bulk cultures. The specificity of hybridization for cellular RNA was demonstrated by pretreating the cells with the Ca2+-dependent enzyme micrococcal nuclease by using a novel protocol developed for use with riboprobes. Maximal levels of granulocyte-macrophage (GM) and multipotential-CSF (interleukin 3) mRNA were detected after 8-10 h, with GM-CSF mRNA being detected earlier and at a lower concentration of stimulus. The rise in intracellular mRNA was accompanied by an increase in the corresponding CSF bioactivity in the supernatant. In situ hybridization was of comparable sensitivity to Northern blot analysis and revealed significant heterogeneity in the accumulation of CSF mRNA within individual cells of the clone following stimulation with F23.1. This could account for the corresponding heterogeneity in CSF production by single cells. Under optimal conditions at least 25% of cells contained both transcripts. The method has been used to examine CSF production by normal spleen cells and should be useful in the further analysis of lymphokine gene regulation in single T cells.
Williamson
D J
DJ
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Owens
T
T
Pearse
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D001345:Autoradiography; D015152:Blotting, Northern; D002460:Cell Line; D003115:Colony-Stimulating Factors / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008808:Mice, Inbred CBA; D008811:Mice, Inbred DBA; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000378:metabolism*; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003115:Colony-Stimulating Factors; D012333:RNA, Messenger