Polystyrene Microsphere Conjugation Protocol

Polystyrene microspheres from Bangs Laboratories (Fishers, IN) with a mean diameter of 10 μm were conjugated using a technique previously described^{51 (link)}. In brief, microspheres were washed in TBS pH = 4.0, twice in DPBS, and then incubated at 2.5 × 10⁷ microspheres/mL in the desired molecular probe in DPBS at equimolar concentrations (relative to 10 ug/mL of recombinant human P-selectin Fc chimera). Conjugated microspheres were washed then blocked with 1% BSA,1% FBS in DPBS. Immediately prior to conducting DBTA, microsphere conjugation was verified via flow cytometry where 100,000 microsphere samples were incubated with the appropriate fluorophore-conjugated antibody or control (e.g., PE-conjugated mouse anti-human CD62P or PE-conjugated mouse IgG1) at 5 µg/mL concentration in 0.1% BSA, DPBS for 30 min. These microspheres were washed twice with 1% BSA and once in DPBS then resuspended in DPBS and analyzed by a FACSAria Special Order Research Product flow cytometer/sorter (BD Biosciences, San Joes, CA). Prior to perfusion in the dynamic biochemical tissue analysis, DBTA probes (conjugated microspheres) were resuspended, unless noted otherwise, at a concentration of 5 × 10⁵ probes/mL in DPBS and verified with a Scepter 2.0 handheld, automated cell counter (EMD Millipore).