Spectramax 250 microplate reader

Anti-proliferative activity in compounds 4a–q were tested in three cancerous cell lines (MCF-7 breast cancer cells, A549 lung carcinoma, HCT human colon cancer cells) and in WI-38 human lung fibroblast cells. Cells were plated at a density of 1.2–1.8 × 10,000 cells/well in a volume of 100 µL complete growth medium and were incubated at 37 °C for 24 h. Cells were then treated with serially diluted tested compound and incubated at 37 °C for 48 h. Twenty microliters of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (2.5 mg/mL) dissolved in PBS were added to the cells and cells were further incubated for 4 h at 37 °C. To dissolve formazan crystals, MTT solutions were completely removed and 100 μL DMSO was added. The absorbance was detected at 540 nm using a Spectramax 250 microplate reader (Molecular device, San Jose, CA, USA), and viability (%) was calculated as [optical density (OD) of treated group/OD of control group] × 100.

hPSC-RPE cells were cultured on Transwell membranes (0.33 cm², Millipore) coated with different substrates. Supernatants from both the hPSC-RPE apical and basal sides (meaning upper and lower compartments of the transwell, respectively) were collected 60 h after the medium was changed. PEDF secretion levels were measured in triplicates for each condition with commercially available human PEDF ELISA Kits (BioVendor RD191114200R) were used, in accordance with the manufacturer’s instructions, after 60 days of culture. The optical density Sreadings were measured using SpectraMax 250 Microplate Reader (Molecular Devices). Results are presented as mean ± SEM.

Total phenolic compounds (TPC) in all leaf extracts were determined spectrophotometrically using the Folin–Ciocalteu method [45 ]. Briefly, 12.5 µL of each extract was taken in triplicate and mixed with 32 µL of Folin–Ciocalteu reagent (1N) and 156 µL of 20% NaCo₃. The resulting mixture was incubated in the absence of light for 2 h at room temperature. Subsequently, the absorbance was read at a wavelength of 750 nm in a Spectramax 250 Microplate Reader (Molecular Devices, Marshall Scientifica, Sunnyvale, CA, USA). A calibration curve was prepared using gallic acid as a standard. TPC was reported as mg gallic acid equivalents per 100 g of dry matter (mg GAE/100 g DM).

From samples of acclimated exponential phase M. tundripaludum SV96^T cultures (75 ml) with densities of ~1–3 × 10⁷ cells mL⁻¹ 22 mL were transferred to three 125 mL glass bottles per temperature. Bottles were then prepared with headspaces of 80% air and 20% CH₄ and a pressure of 1.3 atm. This resulted in 0.36–0.51 mM dissolved CH₄, depending on the temperature, above the threshold of CH₄ saturation for M. tundripaludum SV96^T at this density (0.1 mM CH₄; Fig. S4B). The bottles were incubated at 8, 15, 21, and 27 °C and 50 rpm (Fig. S2A) or 150 rpm (Fig. S2E) for up to 36 h. See SD B: Table B11, B16 for incubation times and sampling time points for each experiment. At time zero and respective sampling intervals, 300 µL of cell suspension (in duplicate) was transferred to a Nunclon Delta Surface plate (Thermo Scientific, Waltham, MA, USA) and the optical density was measured (Spectra Max 250 microplate reader, Molecular Devices, San José, CA, USA) at 600 nm (diluted NMS medium as blank). For optical density measurements (OD₆₀₀), blanks were subtracted from measurements.

Chromogenic substrate pGlu-Phe-Lys-pNA (S-2403) was from Chromogenix (Sweden). 4-Nitrophenyl 4-guanidinobenzoate hydrochloride (pNPGB) was from Aldrich. NUPAGE 4–12% BT GEL was from Invitrogen. Other chemicals and protein reagents were from SIGMA/Aldrich. Kinetic measurement was performed similarly as described^{47 (link)}. Briefly, the refolded and purified μPlg zymogens (35.5 µM) were activated with a plasminogen activator such as urokinase (20:1) at 37 °C for 4 min in a reaction mixture containing 25 mM Tris–HCl, pH 7.4, 50 mM NaCl. The active site of the activated μPlm was titrated using pNPGB as described^{52 (link)}. The activated zymogens were diluted to 5.5 µM, and then 10 µl was mixed with 100 µl of 0.0625 mM, 0.125 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1.0 mM, 1.5 mM, or 2.0 mM of substrate S-2403 in the assay buffer (25 mM Tris–HCl, 50 mM NaCl, pH 7.4). The generation of amidolytic activity was monitored (at 405 nm) at 37 °C in 10 s intervals for 20 min using SpectraMax 250 microplate reader (Molecular Devices). The data was plotted as velocity vs. substrate using GraFit version 7 (Erithacus Software) and the V_max and K_m of the wild-type and each mutant µPlm were determined. The catalytic efficiency (Kcat/Km) was calculated according to the active enzyme concentration.

Cells were seeded onto 96‐well plates (1 × 10³/well) and, after an overnight attachment period, were exposed to the various concentrations of appropriate drugs for 72 h. Cell viability was analyzed using Cell Titer Glo (Promega) according to manufacturer's protocol. The survival curves were calculated by Prism 5 software. For colony formation assays, siRNA‐transfected single cells were seeded onto 6‐well plates (3 × 10³/well) and, after an overnight attachment period, were exposed to the appropriate drugs for 7 days. Media including the corresponding concentration of drugs were replaced every 3 days. Upon treatment completion, cells were washed with phosphate‐buffered saline (PBS), fixed in 4% paraformaldehyde for 10 min, and stained with 0.5% crystal violet for 20 min. To evaluate clonogenicity, images were captured using flatbed scanner and then the cells were dissolved with 20% SDS. The OD of the colony formation was read at 570 nm using a SpectraMax 250 microplate reader (Molecular Devices).