Elisas

Liver function was assessed by analyzing serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. ALT and AST levels were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the manufacturer’s instructions.
The levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis alpha (TNF-α) were measured in serum samples using commercially available enzyme-linked immunosorbent assays (ELISAs) (RayBiotech) according to the manufacturer’s instructions.

The content of angiogenic growth factors in samples of processed, dehydrated human amnion/chorion grafts (dHACM) from eight donors was measured with standard enzyme-linked immunosorbent assays (ELISAs; RayBiotech, Inc., Norcross, GA). Weighed, minced dHACM samples were placed in lysis buffer containing protease inhibitors for 24 hours at 4°C. Samples were then homogenized, centrifuged to remove tissue residue, and the amount of specific angiogenic factors in the lysis buffer was measured in diluted aliquots with standard ELISAs. Growth factor content was normalized to the dry mass of starting tissue.

Blood pressure and heart rate was obtained with an automated blood pressure monitor (Omron Healthcare Inc., Milton Keynes, UK) following > 15 min of quiet sitting. For plasma hormone measurements, 12-h fasted venous blood samples (~ 20 mL) were collected into EDTA-coated vacutainer tubes and centrifuged (Hettich Rotina 46R5) for 15 min at 2500 rpm at -4 °C. After separation, plasma was stored at − 80 °C until analyzed. Plasma concentrations of insulin (INS), ghrelin (GRL), and glucagon-like polypeptide -1 (GLP-1) were analyzed using ELISA’s (RayBiotech, Peachtree Corners, GA, USA). Glucagon and insulin-like growth factor -1 (IGF-1) were analyzed by custom-plex immunoassays (Eve Technologies Corporation, Calgary, AB Canada) and, blood glucose and lipids were determined using a colorimetric assay (Cholestech LDX Analyzer, Abbott Laboratories, Abbott Park IL, USA). Test–retest intraclass correlation (r) and coefficient of variation (CV) in our laboratory with n = 15 was: insulin, and glucose (mg/dL) r = 0.95, CV = 3.2%, and r = 0.97, CV = 5.3%, respectively.