Biotek spectrophotometer

A modified, enzyme-linked immunosorbent assay (ELISA)-based LPS binding method was used to detect interactions between LPS and SPLUNC1 as described previously [11 (link)]. Briefly, 96-well plates were coated overnight with purified LPS (400 ng) from H. influenzae and P. aeruginosa strain 10 (MilliporeSigma). Wells were washed and blocked with 1% bovine serum albumin (BSA)– PBS for 1 h, then, 400 ng of purified SPLUNC1 was added to each well in triplicate. PBS was used as a control for this experiment. An antibody specific to human SPLUNC1 (R&D Systems) diluted 1:5000 with BSA, was used to detect the LPS-bound SPLUNC1. Horseradish peroxidase-conjugated anti-goat antibody was used as the secondary antibody to detect binding rSPLUNC1. Enzyme activity was detected using a TMB Ultra 1-step assay (Pierce Biotechnology) and reaction was stopped with H₂SO₄ (Fisher). Absorbance was detected at OD_450nm in a BioTek spectrophotometer (BioTek).

Exposed glass fiber filters were transferred to 15-mL glass tubes and eluted in 5 mL endotoxin-free water containing 0.05% Tween 20 by orbital shaking for 1 h. The filters were then removed from the tube, and the suspension was centrifuged at 1,000 × g for 15 min to pellet the particulate fraction. The supernatant underwent a second centrifugation step before being aliquoted without the pellet and stored at −20°C until analysis. For endotoxin quantification, supernatants were diluted 20 to 50 times before a Limulus amoebocyte (LAL) kinetic-QCL assay was applied according to the manufacturer’s instructions (Lonza Ltd., Basel, CH). The detection limit of the assay was estimated at 0.5 to 2.5 EU/filter depending on the dilution rate. Parallel controls consisting of samples spiked with 10 μL endotoxin solution (50 EU mL⁻¹) and blank samples were run to assess possible inhibition or enhancement of the sample matrix. The endotoxin concentrations were determined by kinetic measurement of absorbance at 405 nm using a BioTek spectrophotometer (BioTek Instruments Inc., VT, USA) in accordance with the five-point standard curve with concentrations ranging from 0.005 to 50 EU mL⁻¹.

The samples were microcentrifuged at 3000 rpm for 10 min and the supernatant (serum) was removed and used for analysis after incubation at room temperature for 1–2 h to allow for clotting. The creatine kinase (CK) assay was carried out using a commercially available kit (CK Cinético Crystal, BioClin, Ireland) and a BioTek Spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). Values are reported as international units per litre.
Enzyme-linked immunosorbent assay(ELISA)The samples were quantified using a Quantikine IL-1β ELISA kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.

The efficacy of antimicrobial agents on attached biofilms were quantified by measuring the total biomass using crystal violet staining before and after antimicrobial treatments. All centrifugation steps were performed at 12,000× g at room temperature for 5 min. At the end of the treatment regimen, culture media was discarded and attached biofilms were collected by adding 500 μL of 1× PBS before being pelleted. Supernatant was discarded and 50 μL of (0.01% w/v) crystal violet (Sigma, St. Louis, MO, USA) was added to biofilms prior to a 10-min incubation at room temperature. Unbound stain was removed by centrifugation before the biofilm pellet was washed with non-sterile 1× PBS and re-centrifuged. The resulting supernatant was discarded and 200 μL of 10% acetic acid (Sigma, St. Louis, MO, USA) was added to the pellet to release and dissolve excess crystal violet stain during a 15-min incubation period at room temperature. Following incubation, the biofilms were centrifuged and the remaining crystal violet staining was extracted, transferred to a 96-well plate and read at 595 nm using a BioTek Spectrophotometer (BioTek, Winooski, VA, USA).