Victor3 1420 multilabel counter

Renal function was assessed by measuring plasma urea concentrations (Urea UV enzymatic, colorimetric kit, Diagnosticum Inc., Budapest, Hungary) according to the manufacturer’s protocol, and optical density was read at 340 nm (Victor3™ 1420 Multilabel Counter (PerkinElmer, Wallac Oy, Turku, Finland)). BUN values were calculated from the urea concentrations.
Plasma Lcn-2 protein concentration was assayed by the mouse Lipocalin-2/NGAL DuoSet ELISA Development kit (R&D Systems, Minneapolis, MI, USA). Briefly, 96-well plates (Nunc™ GmbH & Co. KG, Langenselbold, Germany) were coated with capture antibody, and non-specific binding sites were blocked with reagent diluent. Plasma samples were incubated in duplicates for 2 h, and the detection antibody was added. Next, Streptavidin-HRP was linked to the detection antibody, followed by a short incubation with TMB substrate (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Samples were washed between each step (5 times with 300 μL washing buffer) until the addition of the substrate solution. Optical density was read at 450 nm with wavelength correction set to 544 nm (Victor3™ 1420 Multilabel Counter (PerkinElmer, Wallac Oy, Turku, Finland)). Concentrations were calculated using a four-parameter logistic curve fit.

CellTox Green® cytotoxicity assay (Promega) was employed to evaluate cytotoxicity and used according to the manufacturer's protocol. In brief, 1 × 10⁴ cells/well were plated overnight in a 96-well plate. Cells were treated with PM2.5 (1,000, 5,000 and 10,000 ng/ml) for 48 h. The CellTox green dye was diluted 1/500 in CM and applied to the cells. After 15 min of incubation at RT, fluorescence was measured at 485/535 nm using a VICTOR³ 1420 Multilabel Counter (Perkin-Elmer) plate reader. ROS was measured by ROS-Glo H₂O₂ Assay (Promega) according to the manufacturer's protocol. In short: 1 × 10⁴ cells/well were plated in white, clear-bottom 96-well tissue culture plates, incubated over night to adhere and subsequently exposed to PM2.5 (5,000 and 10,000 ng/ml) for 48 h. The H₂O₂ substrate solution (25 μM) was added to each well and incubated for 6 h at 37°C in a CO₂ incubator. With this, the H₂O₂ substrate reacts directly with H₂O₂ in the cells and generates a luciferin precursor. Thereafter, ROS-Glo Detection Solution was added and incubated for 20 min at 25°C to generate a luminescence signal. Luminescence was measured using a VICTOR³ 1420 Multilabel Counter (Perkin-Elmer) plate reader.

Cell viability was determined with the colorimetric MTT assay. HaCaT cells were seeded onto 96-well plates at a density of 2 × 10⁴ cells/well and allowed to reach confluence over 24 h. Solutions of the extracts were mixed with Hank’s balanced salt solution (HBSS; pH 6.0, Capricorn Scientific, Ebsdorfergrund, Germany). Prior to the treatment with the extracts, the cell culture medium was withdrawn, and the cells washed with HBSS. The cells were then exposed to the solutions of the extracts in concentrations of 2.5–250 μL/mL for 2 h. Cells incubated in HBSS were used as a negative control. After 2 h of treatment with the extracts, the cells were washed twice with HBSS and incubated with fresh medium (500 µL/well) for 24 h. A total of 50 µL of the MTT solution (5 mg/mL) was added to each well. After 1 h at 37 °C, the medium was removed, and the cells were lysed. Formazan was dissolved with acidic isopropanol and its quantity quantified spectrophotometrically at 570 nm (1420 Multilabelcounter VICTOR3, PerkinElmer, Waltham, MA, USA). Metabolic activity was expressed as relative to control (untreated cells incubated in HBSS).