In situ hybridization of immunoglobulin-specific RNA in single cells of the B lymphocyte lineage with radiolabelled DNA probes.
A method for in situ hybridization has been developed which detects immunoglobulin-specific mRNA transcripts in single murine B lymphocytes with radiolabelled, immunoglobulin gene-specific single-stranded DNA probes. The method has been applied to myeloma and hybridoma cells and to B lymphocytes at various stages of their maturation from small, resting B cells to Ig-secreting plasma cells. A critical step in the procedure is the treatment of the cells with pronase. The various cell types have been found to be differently susceptible to this treatment. Single-stranded DNA probes of different lengths, i.e., between 26 and 1000 bp, have been employed in the hybridization. The number of silver grains over a cell increases proportionally with the length of the probe and with its concentration in the hybridization reaction. The kinetics of the increase of mu-heavy chain-specific RNA molecules in single cells and the appearance of 'switched', gamma-heavy chain-expressing cells are shown after stimulation of murine B cells with lipopolysaccharide.
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D004262:DNA Restriction Enzymes; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007148:Immunoglobulin mu-Chains / Q000235:genetics; D007136:Immunoglobulins / Q000235:genetics*; D007700:Kinetics; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000235:genetics*; D013154:Spleen / Q000276:immunology; D014158:Transcription, Genetic
D007143:Immunoglobulin Heavy Chains; D007148:Immunoglobulin mu-Chains; D007136:Immunoglobulins; D012333:RNA, Messenger; D004262:DNA Restriction Enzymes