In situ hybridization of immunoglobulin-specific RNA in single cells of the B lymphocyte lineage with radiolabelled DNA probes
Berger, C.N.
EMBO Journal 5(1): 85-93
1986
ISSN/ISBN: 0261-4189 PMID: 3007120 Document Number: 282811
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 muturation 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 increase 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.