Supplementary Methods
Flow cytometry. Cell suspensions of spleen were prepared by sieving and gentle pipetting. Bone marrow samples were prepared from femora and tibiae of donor mice.
For surface staining, cells were maintained in the dark at 4oC throughout. Cells were washed twice in ice-cold FACS buffer (2%FCS, 0.02% NaN3 in PBS), then incubated with each antibody and conjugate layer for 30 min and washed thoroughly with FACS buffer between each layer. The following antibodies were used (all from BD except where otherwise indicated): CD3-FITC, CD4-PerCP, CD4-APC, CD4-FITC, CD8-
FITC, CD8-PE (Caltag), CD8-PerCP, CD11b-FITC (Caltag), CD11b-biotin (Caltag),
CD11c-APC, Gr-1-FITC (Caltag), ICOS-PE (e-Bioscience), CD44-FITC, CD44-PE,
CD44-APC, CD62L-biotin, CD21-FITC, IgM-APC, IgM-PE, IgM-FITC, IgD-FITC,
CD23-PE, B220-PerCP, B220-APC, B220-PE, B220-biotin, CD19-PE, AA4.1-FITC,
CD19-PE, CD25-APC, CD69-FITC, CD69-biotin, CD86-PE, GL-7-FITC, CD90-PE,
CD40-L biotin, Fas-L-biotin, purified rat-anti-mouse CXCR5, purified rat-anti-mouse
CD200, PDCD1-PE, rat-anti-mouse 1G12 (kind gift from E. Unanue and D.
Peterson), H-2k-FITC, H-2b-PE, 7AAD (Molecular Probes), Ly9.1-biotin, Ly5a-PE,
Ly5a-biotin, Ly5b-FITC. Secondary antibodies and streptavidin (SA) conjugates were: rat-anti-mouse IgG1-APC, rabbit anti-rat-IgG-biotin (DAKO), SA-PE, SA-APC and SA-Cy-chrome. For the CXCR5 and CD200 stains, the secondary antibody was preabsorbed with normal mouse serum and cells were co-stained with hamster anti-
CD3-FITC. For B cell and myeloid cell stains, cells were pre-incubated with Fc receptor blocking antibodies (purified CD16/CD32). Data were acquired on a Facs
Calibur® flow cytometer, and analysed using FlowJo or WinMDI 2.8 software. Cells were sorted on a FACSVantage (BD). Carboxyfluorescein succinamide ester (CFSE) labeling. Cells were suspended at
5.0 x 107/ml in warm RPMI 1640 and incubated for 10 min at 37oC with 5M CFSE
(Molecular Probes). Labelling was quenched with three washes of ice-cold
RPMI/10% FCS.
Immunohistology. Frozen sections of spleen were air-dried and washed in 0.1M
Tris-buffered saline pH 7.6 then primary antibodies were added in TBS and incubated for 45 min. After a further wash in buffered saline, secondary reagents that had been previously absorbed in 10% normal mouse serum were added to the sections for 45 min. The following antibody conjugates were used: sheep-anti-mouse IgD (The
Binding Site) used in conjunction with rat-anti-mouse Syndecan-1 (Serotec) or with rat anti-mouse CD3 (Serotec); rat anti-mouse IgD (Southern Biotech) used in conjunction with PNA-biotin (Vector Laboratories) or with hamster anti-mouse TCR-
chain-biotin. Secondary antibodies used were: rabbit anti-sheep-HRP (Jackson
Immunoresearch), rabbit anti-rat-biotin (Dako) and rabbit anti-rat HRP (Dako). When biotin-conjugated primary or secondary reagents were used, streptavidin alkaline phosphatase (Vector Laboratories) was added after a further wash in buffered saline and incubated for 20 min. Horseradish peroxidase activity was detected using diaminobenzedine tetrahydrochloride solution (Sigma) and hydrogen peroxide.
Alkaline phosphatase activity was detected using the AP-Substrate Kit III (SK-5300,
Vector laboratories) following the manufacturer’s instructions.