A flow cytometry technique to study nuclear factor-kappa B (NFkappaB) translocation during human B cell activation.
We aimed at examining NFkappaB translocation in B lymphocytes during in vitro activation through the specific receptor for antigen using a technique convenient in most laboratories such as flow cytometry. We present here an original, convenient, and reproducible technique to study B cell activation events through NFkappaB translocation by means of a novel, specific flow cytometry assay. Intranuclear translocation of NFkappaB p65 was induced after a 45min stimulation; the highest signal was detected for a 10 ng/ml stimulus compared to the unstimulated condition (P< 0.05). Purified CD19+ B cells--cultured in the presence of optimal concentrations of anti-micro fragment Abs (10ng/ml) for 45min at 37 degrees C--induced a mean 60% (range: 45-67%) MFI-- and thus, nuclear NFkappaB translocation-increase. We observed a one-pike profile of NFkappaB staining in PBMC B cells and a two-pike profile of NFkappaB staining in using tonsil B cells. B cells are susceptible to various dysregulations leading to minor to severe pathology (including immunoproliferative disorders). Studies of signal transduction carried out specifically in human B cells, using a novel technique gave considerable advantages: feasibility, sensitivity, reproducibility, ease.
Cognasse
Fabrice
F
GIMAP-EA3064, Faculté de Médecine, Université Jean Monnet de Saint-Etienne, 15 rue Ambroise Paré, 42023 Saint-Etienne 2, France.
Sabido
Odile
O
Béniguel
Lydie
L
Genin
Christian
C
Garraud
Olivier
O
D018941:Antigens, CD19 / Q000276:immunology; D001402:B-Lymphocytes / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008213:Lymphocyte Activation; D016328:NF-kappa B / Q000378:metabolism*; D014066:Palatine Tonsil / Q000166:cytology; D015398:Signal Transduction
D018941:Antigens, CD19; D016328:NF-kappa B