Vwr uv 1600pc spectrophotometer

The contents of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were determined on day 7 of the salt treatment, as described by Harborne [57 (link)]. Briefly, 100 mg FW of leaf tissue was ground in liquid nitrogen, extracted in 10 mL of 80% (v/v) chilled aqueous acetone, and centrifuged at 7871× g for 5 min at 4 °C. The absorbance was recorded at different wavelengths (470 nm, 663 nm, and 645 nm) using a UV–visible spectrophotometer VWR UV-1600PC Spectrophotometer (VWR International Europe, Leuven, Belgium). The pigment contents were calculated as follows:



A = absorbance, W = fresh weight of sample (mg), and V = volume (L).

Cells were incubated at 1 × 10⁵ cells per well in a 12-well plate at a final volume of 2000 μL. After 24 h incubation, cells were treated with extracts using three different concentrations (10% extract in the media, the IC₅₀ concentration and one concentration below IC₅₀). The supernatant was collected after 24 h incubation with extracts. To obtain the cell homogenate, 2000 μL of fresh media was added to each well, then the plates were frozen and thawed three times to dissociate the cell membranes, and the cell extract was collected for further analysis. Lactate-dehydrogenase (LDH) and catalase (CAT) [76 (link)], from supernatant samples were analyzed. LDH activity was measured at 340 nm using the LDH Activity Kit for in-vitro use (Biomaxima, Lublin, Poland) and the samples were read with a spectrophotometer (Biochemical Systems International, Model 2000 Evolution, Arezzo, Italy). Catalase activity was measured from the supernatant at 240 nm using an UV-Vis VWR UV-1600PC spectrophotometer (VWR, Radnor, PA, USA). Caspase 3 (Casp3) was determined from the cell supernatant using Human Caspase-3 (Activated) ELISA Kit from Invitrogen (Carlsbad, CA, USA) and the plate was read using a TECAN SPARK10M (TECAN, Austria GmbH, Grodig) according to the assay protocol and the results were calculated using Prism 8 software (San Diego, CA, USA).

Melanin was extracted from dried colonies of C. antarcticus grown on different substrata, following the protocol optimized for black fungi [31 (link)]. After the extraction, purified pigments were dissolved in 500 µL of NaOH 1M and its UV-visible spectrum was measured in VWR-UV 1600 PC Spectrophotometer by using M.Wave professional 2.0 software (VWR, Radnor, Pennsylvania, USA). A standard graph for estimation was used and was made using synthetic melanin. NaOH 1M was used as a blank and the instrument was set in a range of 200–800 nm for the analysis. The correlation between absorbance and wavelength was defined. To determine the concentration of extracted melanin, synthetic DHN (1,8-DiHydroxyNaphthalene) melanin (Thermo Fisher Scientific) was prepared in 1M NaOH at concentrations of 500 mg/mL and a standard curve at 650 nm was obtained as reported in [32 (link)].