Anti cd49d pe

For FACS analysis HUVEC were incubated with anti-HLA-DR PE conjugated antibody (BD-Biosciences, Heidelberg, Germany). Anti-CD3/anti-CD28 activated T-cells four color-staining was performed using the following monoclonal antibodies: anti-CD49d-PE, anti-CD29-APC, anti-CD11a-BV421 and anti-CD18-FITC (all purchased from BD Biosciences, Heidelberg, Germany). For each antibody optimal dilutions were assessed by serial dilutions. Stained samples were measured on a BD FACSlyricTM Flow cytometer using both negative and fluorescence-minus-one controls. Compensation was performed with BD compBeads (BD Biosciences, Heidelberg, Germany). Data analysis was performed using FlowJo software version 5.2 (FlowJo LLC, Ashland, USA).

Freshly isolated CLL B cells were cultured with and without MSCs and treated with 5μM kinase inhibitors as above described. Cells (5×10⁵ for each assay) were collected after 48h, leaving intact the adherent layer, and stained with anti-CD49d PE (BD Biosciences), anti-CCR7 FITC (R&D Systems Inc., Minneapolis, MN, USA), anti-CXCR4 PE (R&D Systems Inc.) anti-CXCR3 PE-Cy5.5 (BD Biosciences, Franklin Lakes, NJ, USA), anti-CXCR5 FITC (R&D Systems Inc.), and anti-CD19 APC (BD Biosciences) monoclonal antibodies. Cells were washed with PBS1X and incubated with saturating concentrations of the appropriate antibodies for 15 minutes at room temperature. 20,000 total events were acquired using FACSCanto A (Becton Dickinson) and the data were analysed by FACSDiva 7 software. Samples were gated on intact cells by forward scatter (FSC) vs side scatter (SSC). For analysis, a second gating step on CD19+ cells was used. Here, we used a difference between the Mean Fluorescence Intensity (MFI) of fully-stained samples and the Fluorescence Minus One (FMO) controls.

Fresh leukapheresis products were stained with the following monoclonal antibodies (MoAbs): anti-CXCR4–PE, anti-CD49d–PE, anti-CD11a–PE, anti-CD34–FITC (BD Biosciences, Franklin Lakes, NJ) or corresponding isotype controls. Additionally, PB mononuclear cells (PBMNCs) were isolated by density gradient centrifugation, viably frozen, and stored in liquid nitrogen until use. Thawed PBMNCs were stained with anti-CD34–FITC (BD Biosciences), CD44–FITC, anti-CD38–PE, anti-CD34–PE (Beckman Coulter), and anti-CD133–APC MoAbs (Miltenyi Biotec, Bergisch-Gladbach, Germany). The percentage of cells expressing respective receptors and mean fluorescence intensity values in relation to isotype controls (MFIRs) were analyzed with a flow cytometer and its accompanying software (Gallios and Caluza, respectively, both Beckman Coulter).