Functional properties of CD19+ B lymphocytes positively selected from buffy coats by immunomagnetic separation
S Funderud1, B Erikstein, H C Asheim
1Laboratory for Immunology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo.
Insights
Human B lymphocytes can be isolated with high purity and viability using anti-CD19 magnetic bead selection. This method preserves cell function for critical assays, showing no interference with activation or proliferation.
Area of Science:
- Immunology
- Cell Biology
Background:
- Efficient isolation of pure, viable human B lymphocytes is crucial for immunological research.
- Current methods may impact cell function or require complex procedures.
Purpose of the Study:
- To develop and validate a direct positive selection method for human B lymphocytes using anti-CD19 magnetic beads.
- To assess the purity, viability, and functional integrity of isolated B cells.
Main Methods:
- Positive selection of B lymphocytes from buffy coats using anti-CD19 antibody (AB1) conjugated to magnetic beads.
- Analysis of cell purity, viability, cell cycle status, and activation markers.
- Functional assays to evaluate B cell activation, proliferation, and differentiation post-isolation.
Main Results:
- High purity (>95%) and viability of isolated B lymphocytes were achieved.
- Isolated cells remained in a resting state and were functionally competent for activation and proliferation.
- CD19 antigen expression was temporarily downregulated post-isolation due to cross-linking but did not impede functional responses.
Conclusions:
- Direct positive selection of B lymphocytes via anti-CD19 magnetic beads is an efficient and reliable method.
- The isolation procedure preserves B cell viability and critical functional capacities.
- Isolated CD19+ B cells are suitable for use in various functional assays, including those assessing activation and proliferation.
Abstract:
Here we report that human B lymphocytes can be positively selected directly from buffy coats applying the anti-CD19 antibody AB1 coupled to magnetic beads. This isolation protocol is highly efficient and the isolated cell population is of very high purity and viability. As judged by cell cycle analysis and various parameters for cell activation, the cells are still in a resting state after isolation. Furthermore, different functional assays have shown that the isolation procedure does not interfere with either activation or proliferation/differentiation of CD19 selected cells as compared to negatively isolated cells. As a consequence of cross-linking during the isolation process, the CD19 antigen is temporarily down-regulated as measured by AB1 binding. Despite this decreased expression, monoclonal antibodies to the CD19 antigen nevertheless inhibited anti-mu plus B cell growth factor induced B cell activation as reported also for negatively isolated cells. Taken together, the presented data strongly suggest that B cells isolated through the CD19 antigen can be used in critical functional assays.
More Related Videos
09:25Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function
Published on: February 16, 2017
09:07Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations
Published on: August 22, 2019