M5284

Flow cytometry was used to determine the cell surface expression of CAR, αv integrin subunit, and integrin heterodimers αvβ3 and αvβ5. Adherent cells, cultured up to 80% of confluence, were detached by trypsin (Sigma-Aldrich, USA) and washed twice with PBS. For each sample 5 × 10⁵ cells were used. Subsequently, the cells were incubated on ice for 1 h with the specific unlabeled primary antibodies that recognized CAR (05-644 RmcB, Merck Milipore, Germany), αv integrin subunit (407286 272-17E6, Merck Milipore, Germany), integrin heterodimers αvβ3 (MAB1976 LM609, Merck Milipore, Germany) and αvβ5 (MAB1961 P1F6, Merck Milipore, Germany) and isotype control (M5284, Sigma-Aldrich, USA), while its binding was revealed by incubation on ice for 1 h with FITC-conjugated anti-mouse antibody (554001, BD Biosciences, USA) as a secondary reagent. Flow cytometry was performed on FACSCalibur (BD Biosciences, USA), while cell acquisition was made using BD CellQuest software package (BD Biosciences, USA). Data were analyzed using FCS Express 3 (De Novo Software, USA), and showed as transduction efficiency corresponding to the geometric MFI shown as an absolute or relative value.

Monoclonal antibodies recognizing human OPN were produced as previously described [20 (link)]. Antibody 9–3 was raised against peptide C-GDSVVYG while antibody 21–5 was raised against peptide C-TYDGRGDSVVYG-CO-NH₂.
An IgG1 isotype control was used (M5284, Sigma) for in vitro experiments.

The control samples from the inhibition assay were stained to confirm by immunofluorescence microscopy the binding of the CD18a antibody onto the surface of the neutrophils. The permeabilization and the blocking for the staining were performed as above. Myeloperoxidase was detected using a polyclonal rabbit anti-human myeloperoxidase (A039829-2, 3.3 mg, 1:309; Agilent, Santa Clara, CA, USA). For the isotype controls, a rabbit IgG (from rabbit serum, I5006, 1.16 mg, 1:108.75; Sigma-Aldrich GmbH,) and a IgG1 Isotype Control from murine myeloma (M5284, 0.2 mg, 1:200; Sigma-Aldrich GmbH) were used. The first antibodies were incubated for 1 h at room temperature. As secondary antibodies, a goat anti-mouse IgG (H + L) DyLight 633 (#35512, 1:500; Invitrogen) and a goat anti-rabbit IgG (H + L) cross-adsorbed Alexa Fluor 488 (A11008, 1:500; Invitrogen) were used. The secondary antibodies were incubated for 1 h in the dark and at room temperature. The processing of the samples was carried out as explained above. The images were taken using a Leica TCS SP5 AOBS confocal inverted-base fluorescence microscope with an HCX PL APO 40× 0.75–1.25 oil immersion objective (see Figure A5, Appendix A).