Sircol

Acid-pepsin soluble collagen, sulfated GAG (s-GAG), and elastin content were quantified using the Sircol, Blyscan, and Fastin quantitative dye-binding assay kits, respectively (Biocolor, Belfast, UK). Samples were weighed before and after lyophilization (wet weight and dry weight, respectively). Samples were digested in 1% (w/v) pepsin in 0.5M acetic acid (Sigma-Aldrich, St. Louis, MO) for 24 h at 4°C for collagen quantification, 1% papain (Sigma-Aldrich, St. Louis, MO) for 15 h at 60°C for s-GAG content, and in 0.25M of oxalic acid at 100°C for 1 h for elastin assessment. Digests were then quantified using manufacturers’ protocols.

The collagen and sulfated glycosaminoglycan (sGAG) contents of acellular nerve were determined using Sircol and Blyscan assay kits (Biocolor Ltd, UK), respectively, performed according to manufacturer’s instructions (Roosens et al., 2016 (link); Medberry et al., 2013 (link)). For the collagen assay, nerve tissue was digested at 20 mg/mL in 1 mg/mL pepsin for 64 hours at room temperature, and for the sGAG assay, tissue was digested at 20 mg/mL in a solution of 0.1 mg/mL papain (Sigma) (containing 0.2 M monobasic sodium phosphate, 0.1 M sodium acetate, 5 μM EDTA, and 5 μM cysteine) for 18 hours at 65 °C. sGAG content was measured both before and after chondroitinase ABC treatment to distinguish residual heparin/keratin sulfates from chondroitin/dermatan sulfate proteoglycans. All data were normalized to the dry weight of the tissue and are represented in units of mg/mg (n = 11 in duplicate for collagen content, and n=5 in duplicate for sGAG content). Additionally, tissues from age- and sex-matched animals were also normalized to tissue length to account for mass changes during decellularization that may skew the results. These data are represented in units of μg/mm (n = 5 in duplicate for both assays).

Cultures were analyzed for ECM formation, represented by GAG and collagen contents that were normalized by DNA content. For DNA, samples were digested overnight at 65°C in papain extraction reagent (0.1 mg/mL papain (≥16units/mg protein) in 0.2 M sodium-phosphate/ethylenediaminetetraacetic acid (EDTA) aqueous buffer suspension). DNA was quantified using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen), following manufacturer’s instructions. Fluorescence was read at an excitation and emission wavelengths of 480 nm and 520 nm respectively using Cytation-5 Microplate-Reader (Biotek instruments, Vermont, USA). Total GAG and collagen were quantified with Blyscan and Sircol assays (Biocolor Ltd., UK), respectively, per manufacturer’s protocols with the slight modification of adding dyes directly to wells. GAG and collagen absorbances were measured at 656 and 555 nm respectively using a microplate-reader. GAG and collagen contents in μg were normalized by DNA for reported values.

The presence of residual cellular antigens was evaluated by labeling against neurofilament (1:500, RT-97, Developmental Hybridoma Studies Bank, Iowa City, IA) for neurons and S-100 protein (1:400, Z0311, Dako, Santa Clara, CA) for Schwann cells (n≥3). Tissue structure and preservation were assessed using an antibody against laminin (1:500, L9393, Sigma). Immunohistochemistry was performed as described in 2.2 above for caspase-3. Residual DNA was quantified using a Quant-iT™ PicoGreen™ dsDNA assay (Invitrogen), and collagen and sulfated GAG content were quantified using Sircol and Blyscan kits, respectively (Biocolor, Ltd; Carrickfergus, UK). Each procedure was conducted according to the manufacturer’s instructions. Unprocessed nerve samples were used as controls.