A flow cytometry technique to study nuclear factor-kappa B (NFkappaB) translocation during human B cell activation
Fabrice Cognasse1, Odile Sabido, Lydie Béniguel
1GIMAP-EA3064, Faculté de Médecine, Université Jean Monnet de Saint-Etienne, 15 rue Ambroise Paré, 42023 Saint-Etienne 2, France.
Insights
We developed a novel flow cytometry assay to track nuclear factor kappa B (NFkappaB) translocation in activated B lymphocytes. This reproducible method offers a sensitive and easy way to study B cell signaling pathways.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- B lymphocytes play a crucial role in adaptive immunity.
- Dysregulation of B cell signaling can lead to various pathologies, including immunoproliferative disorders.
- Understanding B cell activation pathways, such as NFkappaB translocation, is vital for disease research.
Purpose of the Study:
- To present an original, convenient, and reproducible flow cytometry technique for studying NFkappaB translocation in activated B cells.
- To quantify NFkappaB p65 intranuclear translocation during in vitro B cell activation.
- To establish a sensitive assay for B cell signaling studies.
Main Methods:
- Utilized flow cytometry to detect NFkappaB p65 translocation in purified CD19+ B cells.
- Stimulated B cells with anti-IgM antibodies (10 ng/ml) for 45 minutes at 37°C.
- Analyzed NFkappaB staining profiles in peripheral blood mononuclear cell (PBMC) B cells and tonsil B cells.
Main Results:
- A novel flow cytometry assay successfully detected NFkappaB p65 intranuclear translocation upon B cell activation.
- Optimal stimulation (10 ng/ml) for 45 minutes induced a significant increase in NFkappaB translocation (P<0.05).
- Observed distinct NFkappaB staining profiles (one-pike vs. two-pike) in PBMC B cells versus tonsil B cells.
Conclusions:
- The developed flow cytometry assay is a feasible, sensitive, and reproducible method for studying B cell activation via NFkappaB translocation.
- This technique provides valuable insights into B cell signal transduction pathways.
- The assay facilitates research into B cell dysregulations and associated pathologies.
Abstract:
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.
