Elx808 ultra microplate reader

The activity of Trn αβ liposomes was evaluated following the methodology previously described for thuricin CD. Briefly, the liposome suspension was filtered with a 0.45 μm PES sterile filter. Different volumes of the sterile liposome suspension were added to reach final concentrations of Trn α and Trn β of 1.25 and 2.50 μg/mL in each well. Samples were tested in triplicate. A volume of 50 μL was reached in each well by adding PBS. 150 μL of 10⁵ CFU/mL L. monocytogenes ATCC 1916 culture was added to the wells with sample. Blanks were set up in triplicate with only media, the corresponding blank liposome suspension volume, and PBS. Controls contained inoculated media and PBS. The plate was incubated in a BioTek ELx808 Ultra microplate reader at 37 °C for 24 h. Readings were taken every 30 min at 590 nm. The same procedure was followed for free thuricin (freeze dried thuricin powder directly suspended in acetate buffer).

MTT assay was used to measure cell viability in cultures. Cells were plated at a density of 4000 cells/mL in a 96-well cell culture plate (Corning, Denmark). After administration, 20 μl of .5 mg/ml of MTT (Solarbio, Beijing, China) was added to each well and cells were incubated at 37°C for 4 h. Following that, 150 μl of dimethyl sulfoxide (DMSO) per well was added to solubilize the formazan crystals in viable cells. Optical density (OD) in each well was quantified at 490 nm wavelength using an ELx808 Ultra Microplate Reader (BioTek, Winooski, VT, United States).

Serum samples were assayed using standard commercially available ELISA kits for Superoxide Dismutase (SOD Activity Assay kit), Total Antioxidant Status (TAS, Antioxidant Assay kit), Thiobarbituric Acid Reactive Substance (TBARS, Malondialdehyde-MDA, TCA method kit) (Cayman Chemical Company, Ann Arbor, MI, USA), and Nitrotyrosine (ALPCO Diagnostics, Salem, NH, USA). Serum concentrations for SOD and Nitrotyrosine were determined calorimetrically using a BioTek ELX-808 Ultramicroplate reader (BioTek Instruments Inc., Winooski, VT, USA) at an optical density of 450 nm against a known standard curve using standard procedures, while TAS serum concentrations were analyzed calorimetrically at 405 nm. Lastly, serum concentrations for TBARS were determined fluorometrically using a SpectraMax Gemini multimode plate reader (Molecular Devices LLC, Sunnyvale, CA, USA) at an excitation wavelength of 530 nm and an emission wavelength of 550 nm against a known standard curve using standard procedures. Samples were run in duplicate according to standard procedures. Test to test variability of performing these assays yielded average C_V values for the aforementioned markers of: SOD (±8.35 %), TAS (±14.24 %), TBARS (±8.30 %), and NT (±10.03 %) with a test retest correlation for the same markers of: SOD (r = 0.83), TAS (r = 0.85), TBARS (r = 0.94), and NT (r = 0.99).

Cell proliferation was measured via the “Cell Proliferation Kit II (XTT)” (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, material specimens were placed into wells of 12-well plates and were seeded by 6x10⁴ cells in 1 ml medium for 24 h. Cells normally grown on the plastic cell culture plate served as positive controls. Cells grown on RM-A served as negative controls. Thereafter, 500 μl of the XTT labeling mixture was added to the cells for another 4 h. Quantification was performed by measuring the absorbance of 100 μl aliquots in a new 96-well plate by an Elx808 Ultra Microplate Reader (Bio-Tek Instruments GmbH, Bad Friedrichshall, Germany) with filters for 450 and 650 nm (reference wavelength). Results are shown in arbitrary units. Indirect tests were performed by specimen incubation in 1,8 ml medium for 72 h. This conditioned medium was added to 1x10⁴ L929-cells grown for 24 h in wells of 96-well plates. After additional 24 h proliferation was measured according to the manufacturer’s instructions.