Cxcl12

Namalwa cells pretreated with the indicated concentrations of Btk inhibitors or vehicle were stimulated with anti-IgM or CXCL12 for 30 mins, and then cells were seeded into 96-well plates that were coated with fibronectin (BD Biosciences) or VCAM-1 (Sigma). Thirty minutes later tumor cells were washed twice with PBS and adherent cells were measured using the Cell Titer-Glo luminescent cell viability assay system. Migration assays were performed in triplicate with transwell insert chambers coated with VCAM-1. Namalwa cells treated with Btk inhibitors or not in the upper chamber were allowed to migrate towards lower compartment contained CXCL12 (R&D Systems) for 4 hours. The migration of control untreated cells in the presence of CXCL12 was normalized to 100%.

Lentivirus green fluorescent protein transduced HUVECs (HUVECs-GFP) and lentivirus discosoma sp. Red fluorescent protein transduced pericytes (pericytes-DsRED) were mixed at 5:1 ratio in co-culture medium, which is basal EBM medium supplied with 2% FCS, rhFGF-B, ascorbic acid and 100 UmL^-1 PS. Cell mixture supplied with growth factors, including IL-3, SCF-1 and CXCL12 (BD Bioscience), at the volume of 300 µL was added to 200 µL bovine collagen type I (Gibco). NaOH was used to adjust pH to 7.5. Cell-collagen mixture was added to 96-well plate (50 µL per well). After 1 h incubation, 100 µL EGM2 was added per well and the plate was incubated overnight. The following day IS or control buffer was added to the cells. Images were taken from day 1 till day 3 after stimulation using inverted fluorescence microscope and analysed by AngioSys 2.0. To correct for batch effects, an additional condition of cells cultured in the standard co-culture medium was included in each independent experiment.

Femur samples were fixed in 4% paraformaldehyde (Sigma) for 4 h and equilibrated in 30% sucrose (Sigma)/PBS. After two rinses with cold PBS, samples were decalcified with 0.25 M EDTA (Acros) for 2 days and then embedded in O.C.T. (Sakura). For staining, monoclonal antibodies against VCAM-1 (BD Pharmingen) and CXCL-12 (BD Pharmingen), polyclonal antibodies against IL-7 (Abcam) and IL-15 (Abcam), and cell labeling reagents (Molecular Probes), including 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (5(6)-CFDA, SE; CFSE), CellTracker™ Red CMTPX Dye, and DAPI (4′,6-diamidino-2-phenylindole), were used. For secondary antibodies, Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 488 goat anti-rat were purchased from Abcam. Cryostat sections were carried out in Leica CM1900.