Recombinant human gcsf

Human ARPE-19 cell line and primary RPE cells were from ATCC (Manassas, VA) and Lonza (Walkersville, MD), respectively. Dulbecco’s Modified Eagle’s medium (DMEM), Ham’s/F12 medium, HEPES buffer, phosphate-buffered saline (PBS), penicillin-streptomycin, fetal bovine serum (FBS), 0.05% trypsin/EDTA, recombinant human CTGF, and Alexa Fluor-conjugated secondary IgG were from Invitrogen (Carlsbad, CA, USA). Recombinant human EGF, FGF-2, HGF, IGF-1, IL-6, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PTX3, and VEGF were from R & D Systems (Minneapolis, MN). Recombinant human G-CSF, IFN-γ, MCP-1, TGF-α, TGF-β1, TGF-β2, and TGF-β3 were from PeproTech (Rocky Hill, NJ). Hyaluronidase, protease inhibitors, bovine serum albumin (BSA), paraformaldehyde, methanol, Triton X-100, and Hoechst 33342 dye were from Sigma (St Louis, MO). α-SMA and BrdU antibodies were from Abcam (La Jolla, CA); β-catenin antibody was from BD Biosciences (San Jose, CA); Antibodies to lymphoid enhancer factor 1 (LEF1) and phospho-Smad2/3 were from Cell Signaling Technology (Danvers, MA). HMW HA, Healon^® (~4,000 kDa, medical grade), was from Advanced Medical Optics (Santa Ana, CA). BCA Protein Assay Kit was from Pierce (Rockford, IL). HA quantitation kit was from Corgenix (Broomfield, CO). Plastic culture dishes were from Becton Dickinson (Lincoln Park, NJ).

To quantify G‐CSF release from HA cryogels, recombinant human G‐CSF (300–23, Peprotech, lots: 041777 and 041877) was reacted with sulfo‐Cy5 NHS ester (13,320, Lumiprobe, lot 7FM7C) at a 1:250:25 molar ratio of G‐CSF:EDC:sulfo‐Cy5 NHS ester in MES buffer to form Cy5 G‐CSF. Unreacted EDC and sulfo‐Cy5 NHS ester was removed by overnight dialysis on a 10 kDa dialysis membrane. 1 μg of Cy5 G‐CSF was added to 0.6% wt/vol HA‐Tz solution before mixing with HA‐Nb and cryogelation as described above. To track Cy5‐HA cryogel degradation in mice which received G‐CSF loaded Cy5‐HA cryogels, the same protocol is followed substituting G‐CSF for Cy5 G‐CSF and Cy5‐HA‐Nb for HA‐Nb.