Lobind

All the peptides used in this work were obtained from American Peptide Company (Sunnyvale, CA). For the study of the kinetics of the peptide binding and release, C-terminal FITCtagged lyophilized peptides were purchased from the same company. HA powders (1–3 µm) were provided by Trans-Tech (Adamstown, MD). Rat MSCs were isolated from bone marrow of the rats following our published protocol.^{22 (link)} Low-binding tubes (LoBind, Eppendorf, Hauppauge, NY) and pipette tips (VWR Signature, VWR, Visalia, CA) were employed during the functionalization and binding/release experiments in order to minimize the non-specific peptide adhesion on the plastic surfaces.

Paired normal and iLTS scar fibroblasts were cultured, as described in Gene Expression. Following 72 hours of incubation, one milliliter (mL) of media from each well was transferred to a low-protein binding 1.5 mL microcentrifuge tube (Lo-Bind; Eppendorf, Hamburg, Germany) and processed using the Sircol Soluble Collagen Assay (Biocolor, Carrickfergus, U.K.). Sample absorbance was measured using a BioTek Synergy 2 Microplate Reader (BioTek) at OD of 555 nm.

Band excision was performed in a laminar flow hood while wearing a head covering, a face mask, and extended cuff gloves over the sleeves of a full-length lab coat. To avoid cross-contamination during band excision, lanes from dinosaur, sediment, and blank GuHCl extracts were excised separately, transferred to new sterile Petri dishes, and cut into 10 gel sections, as shown in (Figure 2B). Each section was diced into ~1 mm³ cubes using a new, fresh razor rinsed in Optima-grade methanol (Fisher Scientific), followed by rinsing with Optima-grade water (Fisher Scientific) for each individual gel section. Each diced gel section was then transferred to a new 1.5 mL tube (LoBind, Eppendorf), using tweezers rinsed in a stream of methanol followed by a stream of water before every manipulation. Excised gel bands were then destained using the Pierce Silver Stain Kit for Mass Spectrometry, following manufacturer’s protocols (all buffers made in Optima-grade water and all steps at RT). In brief, pieces were incubated in 200 μL of destain solution (twice for 15 min each, with gentle agitation), then washed in 300 μL of each of the following for 15 min with agitation: 100 mM ammonium bicarbonate in Optima-grade water (ABC); 50% 100 mM ABC, 50% Optima-grade acetonitrile (ACN); and 100% ACN. Dehydrated sections were dried to completion by speed vacuum (~10 min) and stored at −80 °C.