In situ hybridization of immunoglobulin-specific RNA in single cells of the B lymphocyte lineage with radiolabelled DNA probes.

Abstract

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.