Anti sdf 1α

SDF-1 splice variant-specific ELISAs (R&D Systems, Minneapolis, MN) were performed as previously described.^{8 ,21 (link)} Briefly, Tet-Off BMSCs were plated and treated as above. After 24 h, the media were collected and cell lysates prepared in Complete Lysis-M EDTA-free buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN). The anti-SDF-1 capture antibody (R&D Systems) in sodium bicarbonate buffer pH 9.4 was bound to MaxiSorp^™ 96-well plates (Nunc, Thermo Fisher Scientific) overnight. Plates were blocked for 2 h with 1% BSA in PBS. Murine SDF-1α or SDF-1β (PeproTech, Rocky Hill, NJ) standards and samples (1:2 diluted) were incubated for 2 h prior to incubating with the biotinylated anti-SDF-1α and anti-SDF-1β detection antibody (2 h; R&D Systems), respectively. Streptavidin-horseradish peroxidase (R&D Systems) was incubated for 20 min followed by the substrate reagent (R&D Systems) for 20 min. Sulfuric acid (2 N) was added to stop the enzymatic color reaction and absorbance was read at 450 nm. SDF-1α and SDF-1β protein expression was calculated using standard curves and normalized to total protein, which was quantified using the EZQ^® Protein Quantitation Kit (Invitrogen).

Surfaces with uniformly immobilized His-tagged SDF-1α and ICAM-1/Fc were visualized via fluorescence intensity measurement at 532 nm and 635 nm (GenePixPro4200A Microarray Scanner, Molecular Devices, LLC, Sunnyvale, CA) upon binding with fluorescently tagged anti-SDF-1α and anti-ICAM-1 monoclonal antibodies (R&D systems, Minneapolis, MN), respectively. To verify specific binding of His-tagged SDF-1α, after fluorescence scanning, the slide was incubated with 1M Imidazole (Sigma, St. Louis, MO) for 10 mins on a rocker, followed by a deionized water rinse. Imidazole competes with the His-tag of protein for binding with surface-immobilized nickel, displacing the protein [34 (link)]. A second fluorescence intensity measurement of the slide (at the same laser power and gain settings as before) was conducted to investigate reduction in surface fluorescence via displacement of His-tagged protein-antibody complex by Imidazole.