Protocol to measure endotoxin from opaque tissues in mice using an optimized kinetic limulus amebocyte lysate assay

Abstract

Endotoxin accumulation has been widely noted in several pathologies ranging from metabolic dysregulation to bacterial infection. Using limulus amebocyte lysate (LAL) assays to detect endotoxin load has been the only reliable way to assess endotoxin accumulation, but assays optimized for detection in opaque tissues are still lacking. We optimized a sensitive Kinetic LAL assay for endotoxin detection from murine tissues. In this protocol, we describe tissue collection and homogenization, followed by the procedure to run the assay and data analysis. For complete details on the use and execution of this protocol, please refer to Ceasrine et al. (2022).

Keywords: Immunology; Neuroscience.

Graphical abstract

Figure 1

Preparing tubes for freezing samples Creating a small hole in the tubes used for collection prevents tubes from popping during rapid temperature changes.

Figure 2

Sealing glass tubes for sample storage Sealing the tube thoroughly prevents contamination between homogenization and running the assay.

Figure 3

Serial dilution for endotoxin standards The amount of water needed to add to the lyophilized standard to obtain 50 EU/mL is lot dependent. Figure made with BioRender.

Figure 4

Example trace results of kinetic LAL assay Optical density (absorbance at 405 nm) versus time is used to determine the 0.2 ΔO.D. threshold (when the absorbance has changed by ≥0.2 compared to the baseline reading). The time to threshold from this baseline corrected data is then used to extrapolate EU/mL from the standard curve. Example graphs in this figure correspond to example data in Table 1.