Growth and detection of human bone marrow B-lineage colonies.
A reproducible method for growing normal human bone marrow B-lineage colonies in agar is described. The clonogenic cells require a rich medium, Opti-MEM (GIBCO/BRL, Burlington, Ontario, Canada), and a source of T-cell-derived factors for growth. The conditions described appear to be limiting for the colony progenitor, suggesting assay clonality. Three novel methods that permit routine and rapid detection of these human B-cell colonies are also described. Colonies containing cells that secrete immunoglobulin are detected by plaquing and protein immunoblotting, while RNA transcripts can be detected by RNA colony blotting. The detection of more than one secreted immunoglobulin isotype or RNA species in a single colony can also be achieved. This B-cell colony assay and the associated detection methods will allow the further delineation of human B lymphopoiesis in both normal and disease states.
Bioresearch Division, Ontario Cancer Institute, Toronto, Canada.
K
K
McGinnes
E
E
Keystone
E
E
Bogoch
D
D
Hastings
H A
HA
Messner
N
N
Jamal
C J
CJ
Paige
D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology; D001853:Bone Marrow / Q000276:immunology* / Q000473:pathology; D001854:Bone Marrow Cells; D002455:Cell Division; D002460:Cell Line; D002469:Cell Separation; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D007075:Immunoglobulin M / Q000032:analysis; D007145:Immunoglobulin kappa-Chains / Q000032:analysis; D007146:Immunoglobulin lambda-Chains / Q000032:analysis; D010003:Osteoarthritis / Q000473:pathology; D011506:Proteins / Q000032:analysis; D012313:RNA / Q000032:analysis / Q000235:genetics
D007075:Immunoglobulin M; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains; D011506:Proteins; D012313:RNA