Spectramax plus 384

MDCK cells were seeded at a density of 1.47x10³ cells/cm² in 96-well plates. Cellular viability and cytotoxicity were evaluated at 48 and 96 hours after xanthine or uric acid treatments. MTT was given to cells at the final concentration of 0.5 mg/ml. After 4 hours of incubation at 37°C, cell medium was removed and the salt crystals formed inside the cells were solubilized with dimethyl sulfoxide (DMSO). Absorbance was then read at 570 nm using a spectrophotometer (Spectra Max Plus 384, Molecular Devices, Sunnyvale, CA, USA). Cellular cytotoxicity was evaluated by Lactate Dehydrogenase (LDH) assay (Bio Vision Inc., Milpitas, CA). MDCK cells were seeded as in MTT assay protocol and, at the end of the appropriate treatment, 100 μl of cellular supernatant were transferred to corresponding wells in an optical clear 96-well plate. 100 μl of reaction solution, consisting of catalyst solution and dye solution in a ratio 1:45, were then added to each well and incubated for 30 minutes at room temperature, protecting the plate from light. Absorbance was finally measured at 500 nm using a spectrophotometer (Spectra Max Plus 384, Molecular Devices). As positive control, Triton X-100 (1% final concentration) treated cells were considered, whereas free medium was used as negative control.

The enzyme inhibition assay was performed by using 6.2 nM E. coli MurC (UDP-N-acetylmuramic acid:L-alanine ligase) and 196 AM ATP, 75 AM UNAM, and 120 AM L-alanine. For IC₅₀ determinations, Compound 4e was dissolved and serially diluted in dimethyl sulfoxide (DMSO) and 2 μl added to each reaction to span a concentration range from 200 to 0.4 μM. AMP-PCP was dissolved in water and added to each reaction to span a concentration range of 2 mM to 4 μM. Reactions were incubated for 20 min at room temperature and then quenched by addition of 150 μl Malachite green phosphate detection reagent [24 ]. After 5 min, microtiter plates were read for absorbance at 650 nm using a Spectramax 384 Plus reader (Molecular Devices). IC₅₀ values were calculated by fitting to the two-parameter equation for inhibition in GraFit 4.0 [25 ].

The absorbance spectrum of palythine was measured between 280 and 400 nm using a Spectra Max 384 Plus (Molecular Devices, Sunnyvale, CA, U.S.A.) spectrophotometer.

LPL activity assays were performed as previously described [16 (link)]. Briefly, Intralipid (160 µg TG per well) served as the substrate in a reaction volume of 50 µl in phosphate-buffered saline (pH 7.4), containing 0.1% (w/v) fatty acid free bovine serum albumin, and the generation of FFAs after 1 h at room temperature was used as a measure of LPL activity [22 (link)]. A total of 0.2 units of LPL purified from bovine milk (Sigma-Aldrich, St. Louis, MO) was added as well. FFAs generated during the reaction were quantified in the same plate, using commercially available enzymatic reagents (Wako Chemicals, USA). Samples were read at A550 in a SpectraMax 384 Plus plate reader (Molecular Devices, Sunnyvale, CA).

Human keratinocyte cells and HEM cells (2 × 10³ per well) were collected and seeded in triplicate 96-well plates in 100 μl of growth medium. Different seeding densities were optimized at the beginning of the experiments. 24 h after irradiation or SM treatment, Cell Count Kit-8 assay (Dojindo Laboratories, Kumamoto, Japan) was used to assess the cell viability of the HaCaT cells and HEM cells according to the manufacturer’s instructions. Next, the absorbance of each well was measured at an absorbance of 475 nm with the Spectra Max 384 PLUS microplate reader (Molecular Devices, Sunnyvale, CA, United States).