Sodium alginate

Sodium alginate with high (232 kDa) and medium (139 kDa) molecular weight and high guluronic acid content was purchased from FMC Biopolymer (Princeton, NJ). To produce the low molecular weight alginate, the high molecular weight alginate was gamma-irradiated with a cobalt-60 source at a gamma-dose of 3 Mrad. Alginates were covalently coupled with the integrin-binding peptide (Gly)4-Arg-Gly-Ala-Ser-Ser-Lys-Tyr (Peptides International) and either Fluoresceinamine, isomer I (Sigma-Aldrich) or LissamineTM Rhodamine B Ethylenediamine (Setareh Biotech). 1-bicyclo[2.2.1]hept-5-en-2-ylmethanamine (norbornene; Matrix Scientific) or 3-(p-benzylamino)-1,2,4,5-tetrazine (Sigma Aldrich) were prepared and conjugated to 232-kDa alginate as described previously²³ .

All reagents were purchased from Sigma-Aldrich and used without any further purification unless otherwise indicated. C2C12 mouse premyoblasts and Dulbecco's modified Eagle's medium (DMEM) were obtained from ATCC. Foetal bovine serum (FBS), penicillin/streptomycin (P/S), phosphate buffered saline (PBS), Modified Eagle's Medium (MEM), Hank's Balanced Salt Solution (HBSS) were ordered from Gibco® (Life Technologies). Normal goat serum, modified nucleotide 5-ethynyl-2′-deoxyuridine (EdU), 4',6-diamidino-2-phenylindole HCl (DAPI) and all Alexa Fluor® conjugated products for immunofluorescence staining were ordered from Life Technologies. Sodium alginate was purchased from FMC Biopolymer.

Sodium alginate with ~240 kDa molecular weight (LF200) was purchased from FMC Biopolymer. An integrin-binding peptide consisting of Arg-Gly-Asp (GGGGRGDSP; Peptide 2.0) was covalently conjugated to alginate by 1-ethyl-dimethylaminopropyl (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) (Thermo) chemistry with ~60 μM as described previously^{62 (link)}. After conjugation, alginate-RGD was dialysed against decreasing concentrations of NaCl, charcoal-treated, filter-sterilized and lyophilized. Lyophilized alginate was stored in −20 °C and dissolved in DMEM within 1 week before experiments. The amount of RGD conjugated to alginate was quantified by the LavaPep kit (LP-022010, Gel Company) as described^{63 (link)}, which is based on a naturally occurring fluorescent compound that binds to lysine, arginine and histidine residues in peptides^{64 (link)}. After reacting the compound with RGD standard solutions dissolved in unconjugated 1% w/v (41.7 μM) LF200 alginate and 1% w/v alginate-RGD samples for 1 h at 37 °C, fluorescent signals were acquired by FlexStation 3 (Molecular Devices) at excitation/emission = 540/630 nm. To visualize alginate gels by fluorescence, a small amount (final w/v = 0.025% or 2.085 μM) of 10/60 alginate (~120 kDa; FMC Biopolymer) coupled with lissamine rhodamine B ethylenediamine (Thermo) was added before gel formation.