Recombinant human cd44 fc chimera protein

Atomic force microscopy (AFM) was performed using custom CD44-functionalized beads on cantilevers. Briefly, protein G coated polystyrene beads (mean diameter 3.4±0.7 μm, Spherotech, Lake Forest, IL) were treated with recombinant human CD44 Fc chimera protein (1 μg/mL, R&D, Minneapolis, MN), which was covalently crosslinked using bis(sulfosuccinimidyl) suberate (1 mM BS₃, Thermo Fisher Scientific, Waltham, MA). CD44-coated beads were washed in deionized water twice for 2 minutes, plated on glass, dried, and affixed to tipless silicon SPM-sensors (Arrow TL1, nominal spring constant 0.03 N/m, NanoAndMore). Hydrogels fabricated from macromers were probed with these custom CD44 bead tips in indentation testing at 10 μm/s with zero dwell time and 1.2±0.9 μm maximum indentation depth, and the maximum deflection from baseline in the output retraction curves were used to calculate retraction forces on the cantilever.

Protein G coated plates (Thermo Fisher Scientific, Waltham, MA) were treated with recombinant human CD44 Fc chimera protein (1 μg/mL, R&D, Minneapolis, MN) and subsequently covalently crosslinked using bis(sulfosuccinimidyl) suberate (1 mM BS3, Thermo Fisher Scientific, Waltham, MA). Macromers modified with FITC-GCKK peptide (1:1 molar ratio, peptide:macromer) were added to wells at 200 μg/mL (HA backbone concentration, accounting for mass contribution of pendant groups to ensure constant HA molar amounts across groups) in PBS for 30 minutes. Wells were washed twice for 2 minutes each and then analyzed for FITC signal using a plate reader (Infinite M200, Tecan, Männedorf, Switzerland). Values were normalized to the signal for PEG macromers for reporting.