BM and spleen were processed as previously described (Rothaeusler and Baumgarth, 2006 (link)). All buffers used for staining were azide free. Briefly, BM was harvested by injecting staining media through the marrow cavity of a long bone, and a single-cell suspension was made by pipette agitation and filtering through a 70-μm nylon mesh. A single-cell suspension of the spleen was made by grinding the tissue between the frosted ends of two microscope slides and filtered through a 70-μm nylon mesh. All samples were then treated with ACK lysis buffer (Rothaeusler and Baumgarth, 2006 (link)), refiltered through nylon mesh, and suspended in staining media. Peritoneal/pleural cavity cells were obtained using staining media flushed into and then aspirated from the peritoneal and pleural cavities with a glass pipette and bulb or a plastic pipette (Molecular Bio Products, Inc.). Trypan Blue exclusion dye was performed on all samples to identify live cells using a hemocytometer or an automated cell counter (Nexcelom Bioscience). Cells were blocked with anti-FcR (2.4.G2), washed, and stained with fluorescent antibodies (Table S2 ).
PtC-containing liposomes were generously provided by Aaron Kantor (Stanford University, Stanford, CA, USA). Dead cells were identified using Live/Dead Fixable Aqua or Live/Dead Fixable Violet stain (Invitrogen). Fluorescently labeled cells were read on either a four-laser, 22-parameter LSR Fortessa (BD Bioscience), or a five-laser, 30-parameter Symphony (BD Bioscience). FACS-sorting was done using a three-laser FACSAria (BD Bioscience) equipped with a 100-μm nozzle at low pressure to avoid PC death. Data were analyzed using FlowJo software (FlowJo LLC, kind gift of Adam Treister, Gladstone Institute, San Francisco, CA). Peritoneal B-1 cells are identified as Dump− (CD4, CD8a, Nk1.1, F4/80, Gr-1), CD23−, and CD19hi. Splenic CD4+ T cells were identified as Dump− (CD19, CD8a, NK1.1, F4/80, Gr-1, TCRγδ, CD138), CD3+, and CD4+. BM and spleen B-1 cells are identified as Dump−, CD23− C43+, IgD−, IgM+, and CD19hi; and B-1PC are identified as Dump−, CD23− C43+, IgD−, IgM+, CD19lo/− and Blimp-1; and/or CD138+. FACSAria-sorted cells for adoptive transfer had a purity of >98% for peritoneal B-1 cells and peritoneal cells depleted of B-1 cells and> 99% for splenic CD4 T cells.
PtC-containing liposomes were generously provided by Aaron Kantor (Stanford University, Stanford, CA, USA). Dead cells were identified using Live/Dead Fixable Aqua or Live/Dead Fixable Violet stain (Invitrogen). Fluorescently labeled cells were read on either a four-laser, 22-parameter LSR Fortessa (BD Bioscience), or a five-laser, 30-parameter Symphony (BD Bioscience). FACS-sorting was done using a three-laser FACSAria (BD Bioscience) equipped with a 100-μm nozzle at low pressure to avoid PC death. Data were analyzed using FlowJo software (FlowJo LLC, kind gift of Adam Treister, Gladstone Institute, San Francisco, CA). Peritoneal B-1 cells are identified as Dump− (CD4, CD8a, Nk1.1, F4/80, Gr-1), CD23−, and CD19hi. Splenic CD4+ T cells were identified as Dump− (CD19, CD8a, NK1.1, F4/80, Gr-1, TCRγδ, CD138), CD3+, and CD4+. BM and spleen B-1 cells are identified as Dump−, CD23− C43+, IgD−, IgM+, and CD19hi; and B-1PC are identified as Dump−, CD23− C43+, IgD−, IgM+, CD19lo/− and Blimp-1; and/or CD138+. FACSAria-sorted cells for adoptive transfer had a purity of >98% for peritoneal B-1 cells and peritoneal cells depleted of B-1 cells and
