Detection, by in situ hybridization using sulphonated cDNA probe, the specific mRNA for HLA-DR alpha induced in monocyte cell lines by recombinant interferon gamma.
We adopted the nonradioactive method used for blot hybridization for the detection of inducible mRNA for HLA-DR alpha by the in situ hybridization. Unstimulated and interferon gamma stimulated MonoMac6 and U937 human monocytic cell lines were used as target cells. Sulphonation of plasmid pBR322 with HLA-DR alpha cDNA insert (2 x 700 bp, in Pstl restriction site) was performed according to the manufacturer's procedure (SulfoProbe Kit, Sigma). The hybridization signals were detected with mouse monoclonal, anti-sulphonated DNA antibody, followed by immunovisualization with anti-mouse IgG-alkaline phosphatase conjugates. Unstimulated MonoMac6 and U937 cells showed few granular reaction products only in small percentage of cells (1-5%), while in IFN gamma stimulated cells the fine granular immunoenzymatic reaction was observed in the cytoplasm of majority of cells (> 80%). This method seems to be easy and rapid to perform, making it applicable for routine diagnostic purposes in tissue sections and biopsies.
Wieckiewicz
J A
JA
Department of Clinical Immunology and Microbiology, Copernicus Medical Academy, Cracow, Poland.
Jasiński
M
M
Mytar
B
B
Jasińska
A
A
Uracz
W
W
D015139:Blotting, Southern; D002460:Cell Line; D015342:DNA Probes; D015870:Gene Expression / Q000187:drug effects; D006684:HLA-DR Antigens / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D007371:Interferon-gamma / Q000494:pharmacology*; D009000:Monocytes / Q000187:drug effects* / Q000276:immunology* / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity; D013450:Sulfones
D015342:DNA Probes; D006684:HLA-DR Antigens; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013450:Sulfones; D007371:Interferon-gamma