Polarstar galaxy microplate reader

Complete blood count was performed as haematology diagnostics using ProCyte Dx Haematology Analyser (IDEXX, United States). Blood plasma haptoglobin and CRP concentrations were measured using Phase range Haptoglobin Assay Kit (cat. no. TP-801) and Phase porcine CRP Assay Kit (cat. no. TA-901) respectively (Tridelta Development Ltd., Ireland). Haptoglobin analyses were performed using an ADVIA 1800 Clinical Chemistry System autoanalyzer (Siemens Healthineers AG, Germany), while CRP concentrations were determined by solid-phase sandwich immunoassay, and measured using a POLARstar Galaxy microplate reader (BMG Labtech, Germany). The plasma concentration of cytokines (IFN-γ, IL-1β, IL-6, IL-10, IL-12, and TNF-α) were analyzed using methods previously described (33 (link)). Plasma diamine oxidase activity (DAO) was determined by a kinetic-fluorometric method where 1.5 diamino pentane (cadaverine, Sigma C8561) was the substrate and 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) was the profluophore oxidized by the developed hydrogen peroxide. Units were defined as d-emission per min at 590 nm after excitation at 544 nm. Plasma lipopolysaccharide (LPS) concentration was analysed using the Porcine Endotoxin ELISA kit (AMSBIO, United Kingdom). The assays were performed in duplicates.

Cell viability was evaluated by measuring the fluorescence intensity of cells using the alamarBlue viability assay (Invitrogen Life Technologies, Carlsbad, CA, USA) (6 ). LNCaP cells were seeded in 96-well plates (Sumilon, Tokyo, Japan) at a density of 8×10³ cells/well in RPMI-1640 medium supplemented with 10% FBS. On the following day, cells were treated with various concentrations of each compound and the incubation was continued for three days. AlamarBlue solution was subsequently added to the wells and the plates were incubated for 1 h. Next, the fluorescence intensity of the cells was measured using a POLARstar Galaxy microplate reader (BMG Labtech Ltd., Offenburg, Germany) using excitation and emission wavelengths of 544 and 612 nm, respectively.

After 16 h of A. fumigatus biofilm formation, intracellular reactive oxygen species (ROS) levels were measured in A. fumigatus biofilm exposed to P. aeruginosa filtrates, as previously described [11 (link)–16 ]. A. fumigatus biofilms were scraped from the plate and spiked with dihydrorhodamine (DHR) 123 (5.0 μg/ml). After 2 h at room temperature, the cells were washed with PBS, centrifuged at 13 000 x g for 5 minutes, harvested, and observed with a Nikon Microphot SA fluorescence microscope (excitation, 488 nm; emission, 520 nm). For quantitative assays, fluorescence intensity values were recorded by using a POLARstar Galaxy microplate reader (excitation, 488 nm; emission, 520 nm; BMG LABTECH.