Fluoroskan ascent fl microplate reader

Human fetal lung fibroblasts MRC5 transformed with SV-40 (MRC5 SV2, EcACC Cat. No. 84100401) and primary skin fibroblasts from Common Use Center “Biobank” (Research Centre for Medical Genetics, Moscow, Russia) were cultured in DMEM (Dulbecco’s modified Eagle’s) medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 2 mM glutamine and 10% fetal bovine serum (FBS) (HyClone, Logan, UT 84321 USA) and 100 U/mL streptomycin and 100 U/mL penicillin (all from Gibco, USA). Cell viability was measured using the CellTiterBlue^® reagent (Promega, USA) according to the manufacturer’s protocol with Fluoroskan Ascent FL Microplate Reader (Thermo Labsystems, Waltham, MA, USA).

Cells treated with zymosan and medium 199(Gibco, Grand Island, NE, USA) or melatonin (Sigma, St Louis, MO, USA) were incubatedin the presence of a 5µL Fluo-3 AM solution (Sigma, St Louis, MO, USA). The cells were washed (160 × g, 10 min, 4 °C) and resuspended in a HBSS (Hank’s Balanced Salt Solution) containing bovine serum albumin (BSA) and analyzed by Fluoroskan Ascent FL^®Microplate reader (Thermo Scientific, Vantaa, Finland), with the 485 nm excitation and 538 nm emission filters. The results were expressed as the mean fluorescence intensity of Fluo-3 AM. The experiments were performed in duplicate.

Dihydrorhodamine 123 (DHR 123), which fluoresces in the cell after being oxidized by reactive oxygen species, was used to detect OH and H₂O₂. The MN phagocytes were incubated with DHR123 (5 µ/mL; Sigma, St Louis, MO, USA), following the protocol standardized by Radogna et al. [25 (link)]. Fluorescence intensity was measured on a Fluoroskan Ascent FL^®Microplate reader (Thermo Scientific, Vantaa, Finland), with a 485 nm excitation filter and emission filter of 538 nm. The intensity of the fluorescence emitted is proportional to the production of the reactive oxygen species. The results were expressed as the DHR123 mean fluorescence intensity.
The superoxide release was determined by cytochrome C (Sigma, St. Louis, MO, USA) reduction. After the MN cell treatment, the cells were centrifugated and then resuspended in PBS containing 2.6 mM CaCl₂, 2 mM MgCl₂, and cytochrome C (Sigma, St Louis, MO, USA; 2 mg/mL). The suspensions (100 μL) were incubated for 60 min at 37 °C on culture plates. The reaction rates were measured by absorbance at 550 nm, and the results were expressed as nmol/O₂⁻. All the experiments were performed in duplicate.