title: A two-step non-flowcytometry-based naïve B cell isolation method and its application in Staphylococcal enterotoxin B (SEB) presentation.
abstract: BACKGROUND
  To study the role of human naïve B cells in antigen presentation and stimulation to naïve CD4+ T cell, a suitable method to reproducibly isolate sufficient naïve B cells is required.
  
  
  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 naïve B cells were analyzed for phenotypes and for their role in Staphylococcal enterotoxin B (SEB) presentation to naïve CD4+ T cells.
  
  
  RESULTS
  The mean (SD) naïve 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 naïve CD4+ T cell activation by Staphylococcal enterotoxin B (SEB) presentation.
  
  
  CONCLUSIONS
  This two-step non-flow cytometry-based approach improved the purity of isolated naïve 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 naïve B cells can present a super-antigen "SEB" to activate naïve CD4 cells. These methods may thus be useful for further in vitro characterization of human naïve B cells and their roles as antigen presenting cells in various diseases.
authors.affiliation: Division of Allergy and Clinical immunology, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
authors.forename: Kaj
authors.initials: K
authors.lastname: Chokeshai-u-saha
authors.forename: Supranee
authors.initials: S
authors.lastname: Buranapraditkun
authors.forename: Alain
authors.initials: A
authors.lastname: Jacquet
authors.forename: Catherine
authors.initials: C
authors.lastname: Nguyen
authors.forename: Kiat
authors.initials: K
authors.lastname: Ruxrungtham
mesh_terms: D017951:Antigen Presentation / Q000276:immunology*; D000938:Antigen-Presenting Cells / Q000166:cytology / Q000276:immunology*; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D003584:Cytological Techniques / Q000379:methods*; D003713:Dendritic Cells / Q000276:immunology; D004768:Enterotoxins / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology; D009000:Monocytes / Q000276:immunology; D015854:Up-Regulation / Q000276:immunology
chemical_list: D004768:Enterotoxins; C508017:IL2RA protein, human; D053645:Interleukin-2 Receptor alpha Subunit; C031818:enterotoxin B, staphylococcal