A 96-well microplate (Corning Costar, Cambridge, MA) was used in a heating block at 37°C and maintained at this temperature throughout the assay. The absorbencies of endotoxin were individually measured by using an enzyme-linked immunosorbent assay plate-reader (Ultramark; Bio-Rad Laboratories, Inc, Hercules, CA). The Quantitative Chromogenic LAL-1000 test (QCL-1000) LBH589 cost (BioWhittaker, Inc, Walkersville, MD) was used for the quantification of endotoxin in root canal samples. Initially, 50 μL of the blank were used according to the standard endotoxin concentrations (ie, 0.1, 0.25, and 1.0 EU/mL), and 50 μL of the samples was added in
duplicate in the 96-well microplate. This was followed by the addition check details of 50 μL LAL to each well, and the microplate was then briefly shaken. Ten minutes later, 100 μL of substrate solution (prewarmed to 37°C)
was added to each well, always maintaining the same sequence. The plate was mixed and incubated at 37°C for 6 minutes. Next, 100 μL of a stop reagent (acetic acid 25% v/v) was added to each well, and the absorbance (405 nm) was read by using an enzyme-linked immune-sorbent assay plate-reader (Ultramark, Bio-Rad Laboratories). Both test procedure and calculation of endotoxin level were performed according to the manufacturer instructions. A color interference assay was performed in the QCL-1000 test (chromogenic endpoint assay), according to the manufacturer’s instructions, as recommended if 25% acetic acid is used as stop reagent. The chromogenic kinetic test used for the quantification of endotoxin was the KQCL test (BioWhittaker). First, as a parameter for the calculation of the amount of endotoxins in root canal samples, a standard curve was plotted by using Thiamine-diphosphate kinase endotoxins with a known concentration (50 EU/mL) and their dilutions with the following final concentrations: 0.005,
0.05, 0.5, and 5 EU/mL. One hundred microliters of the blank were used according to the standard endotoxin concentrations (ie, 0.005, 0.05, 0.5, and 5 EU/mL), and 100 μL of the samples were added in duplicate in the 96-well microplate with the respective positive product control. All reactions were achieved in duplicate in order to validate the test, and the absorbance (405 nm) was read by using an enzyme-linked immune-sorbent assay plate-reader (Ultramark, Bio-Rad Laboratories). Both test procedure and calculation of endotoxin level were performed following the manufacturer’s instructions. The turbidimetric test, Pyrogent 5000 (BioWhitaker, Inc, Walkersville, MD), was used to measure endotoxin concentrations in the root canals by using the LAL technique.