Identification of hybridomas derived from mouse CD5+ B lymphocytes by fluorescent staining for cytoplasmic CD5...

The production of hybridomas derived from CD5+ B lymphocytes is important for studying the immunoglobulin gene repertoire and antibody specificity of this B-lymphocyte subpopulation. However, hybridomas derived from these lymphocytes invariably lose surface membrane expression of CD5 following hybridization. This impedes the unequivocal assignment of the generated hybridomas to the CD5+ B-lymphocyte subpopulation from unsorted, or partially sorted, cells. Recent studies have shown that mRNA transcripts of the CD5 gene and the protein product can be detected in the cytoplasm of some mouse hybridomas, indicating that although surface expression has been lost, they may have been generated from CD5+ B lymphocytes. These studies, however, have been unable to discount completely the possibility that aberrant cytoplasmic expression of the CD5 gene occurred as a direct consequence of the hybridization process. To confirm that cytoplasmic CD5 expression in the hybridomas is in fact directly related to the B-lymphocyte origin we have generated hybridomas from FACS-sorted CD5+ and CD5- murine splenic B lymphocytes, and determined their surface and cytoplasmic CD5 expression by fluorescence-activated cell sorting. Our findings reveal that all hybridomas derived from CD5+ B lymphocytes (nine hybridomas) were negative for surface but positive for cytoplasmic CD5 expression, whereas all hybridomas derived from CD5- B lymphocytes (nine hybridomas) were negative for both surface and cytoplasmic CD5 expression. This finding shows that staining for cytoplasmic CD5 expression provides an accurate method of determining the cellular origin of murine B-lymphocyte hybridomas.