A two-step non-flowcytometry-based naive B cell isolation method and its application in Staphylococcal enterotoxin B (SEB) presentation

作者：Chokeshai u Saha Kaj; Buranapraditkun Supranee; Jacquet Alain; Nguyen Catherine; Ruxrungtham Kiat^*

来源：Asian Pacific Journal of Allergy and Immunology, 2012, 30(3): 214-223.

• Naive B cell
• antigen presentation
• naive CD4(+) T cell
• resetting
• magnetic cell sorting
• superantigen
• staphylococcal enterotoxin B (SEB)

Background: To study the role of human naive B cells in antigen presentation and stimulation to naive CD4(+)T cell, a suitable method to reproducibly isolate sufficient naive B cells is required. %26lt;br%26gt;Methods: To improve the purity of isolated naive B cells obtained from a conventional one-step magnetic bead method, we added a rosetting step to enrich total B cell isolates from human whole blood samples prior to negative cell sorting by magnetic beads. The acquired naive B cells were analyzed for phenotypes and for their role in Staphylococcal enterotoxin B (SEB) presentation to naive CD4(+)T cells. %26lt;br%26gt;Results: The mean (SD) naive B cell (CD19+/CD27-) purity obtained from this two-step method compared with the one-step method was 97% (1.0) versus 90% (1.2), respectively. This two-step method can be used with a sample of whole blood as small as 10 ml. The isolated naive B cells were phenotypically at a resting state and were able to prime naive CD4(+)T cell activation by Staphylococcal enterotoxin B (SEB) presentation. %26lt;br%26gt;Conclusions: This two-step non-flow cytometry-based approach improved the purity of isolated naive B cells compared with conventional one-step magnetic bead method. It also worked well with a small blood volume. In addition, this study showed that the isolated naive B cells can present a super-antigen %26quot;SEB%26quot; to activate naive CD4 cells. These methods may thus be useful for further in vitro characterization of human naive B cells and their roles as antigen presenting cells in various diseases.

• 出版日期2012-9