Limulus amebocyte lysate (LAL) endotoxin assay

To determine the scaffold endotoxin content, the AXF foams were weighed, sterilized with 1000 ppm peracetic acid for 15 minutes, washed in PBS three times, and air dried for 10 minutes using the protocol developed by Yoganarasimha et al.³² Sterilized AXF foams and unsterile foams were placed into a tissue culture insert inside each well. The inserts containing the foam samples were immersed in PBS for four hours. After immersion, a 50 μl aliquot was extracted from the release media and diluted 20-fold with addition of 1 mL of endotoxin free media from the endotoxin assay kit. After dilution, 50 μl aliquots were taken from the diluted samples and added to a 96-well plate. Next, 50 μl of LAL reagent was added to the sample for a 10-minute incubation at 37 °C. Afterwards, 100 μl of chromogenic substrate was added to the reaction mixture and incubated for six minutes at 37 °C to induce a colorimetric reaction based on protease enzyme activity from the endotoxin. A 50 μl aliquot of 25% acetic acid was added to end the reaction. Samples were gently shaken and subjected to spectrophotometric measurement at 405 nm for quantification of endotoxin levels against a standard curve.