Microplate fluorometer

MPP⁺- and VPA-treatment effect on cell viability was assessed using a cell viability assay kit (Promega, Madison, WI, USA), according to the manufacturer's instructions. The cell viability assay uses the indicator dye resazurin to measure the metabolic capacity of cells and estimate the number of viable cells. After 48 h, 25 µL of treated cell samples was collected, and then 5 µL of cell viability assay reagent per well was added and the plates were incubated at 37°C for 1 h. The fluorescent signal was recorded by a microplate fluorometer (Thermo Fisher Scientific Inc.) at 560/590 nm.

The ferrous iron measurement on cell was performed using calcein-AM method as previously described, with some modifications^{55 (link)}. Briefly, SH-SY5Y cells were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, and cultured at 37 °C under humidified 5% CO₂ atmosphere. Cells were treated with ferric ammonium citrate (FAC) or GSK-J4 or GSK-J4 plus FAC for 12 h in serum-free media. Then they were washed with PBS twice, followed by the addition of calcein-AM (50 nM), and incubated for 30 min at 37 °C. After that, fluorescence was measured using a microplate fluorometer (Thermo Fisher Scientific Inc).

BBH accumulation was measured as described previously with moderate changes (Wang et al., 2009 (link)). Briefly, bacteria were cultured into the exponential growth phase. They were centrifuged (8000×g, 2min), washed twice with 0.01M PBS (pH 7.2) and re-suspended in 1.5 mL PBS to OD_600nm=1.0. BBH was added with a final concentration of 80 mg/L. 50 μM of an efflux pump inhibitor (i.e. CCCP, PAβN and reserpine) was added at an appropriate time (t = 60 min). Assays were performed in a 96-well black plate with a final volume of 100 μL per well. Fluorescence was measured every 5 min for 90 min in a microplate fluorometer (Thermo Scientific, USA) with an excitation wavelength of 355 nm and an emission wavelength of 538 nm.

Attached cells (10,000 cells per well in a 96-well plate) were washed with phosphate-buffered saline (PBS) and incubated for 1 h with cell-permeable 2ʹ,7ʹ-dichlorodihydrofluorescin diacetate (DCFH-DA) (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions. Highly fluorescent DCF, which is produced as a result of oxidation of DCFH by cellular ROS, was measured using a Microplate Fluorometer (Thermo Fisher Scientific).

Harvested cell was subjected to two times of freeze and thaw in 100 μL of PBS. The lysed cell was then pelleted and the supernatant was subjected to GSH and ROS quantification using Glutathione assay kit (Sigma, USA) and OxiSelect ROS assay kit (Cell Biolabs, USA) according to manufacturers’ protocol.
For GSH quantification, 10 μL of cell lysate supernatant was added with 150 μL of working solution (1.5 mg/mL DTNB solution, 6 units/mL glutathione reductase and 1× assay buffer), incubated for 5 min and added with 50 μL of NADPH solution (0.16 mg/mL). The absorbance was read at a wavelength of 412 nm by ELISA plate reader (Bio-Tek instrument, USA) for every minute in duration of 5 min. For intracellular ROS quantification, supernatant was added with 10 μM DCFH-DA for 30 min at 37 °C. Then, the fluorescence intensity of DCFH-DA was measured using microplate fluorometer (Thermo Scientific, USA) with a 485/538 nm filter. Fold change of GSH and ROS was calculated by dividing absorbance or fluorescence intensity of DK1 treated MCF-7 with untreated control MCF-7.

RNA was extracted from papaya flesh using NucleoZOL (Machery-Nagel Inc., Allentown, PA, USA) from approximately 50 mg of fruit flesh per sample according to the manufacturer’s instructions. Total RNA quantity and quality were assessed by a spectrophotometer (Microplate Fluorometer, Thermo Fisher Scientific, Waltham, MA, USA), using light absorbance of RNA at wavelengths of 260 nm (OD₂₆₀) and 280 nm (OD₂₈₀).

A working stock solution of freeze dried venom was reconstituted in a buffer containing 50% MilliQ/50% glycerol (>99.9%, Sigma, St. Louis, MO, USA) at a 1:1 ratio to preserve enzymatic activity and reduce enzyme degradation. Varying concentrations of crude venom (10 ng/µL and 50 ng/µL) were plated out in triplicates on a 384-well plate (black, Lot#1171125, nunc™ Thermo Scientific, Rochester, NY, USA) and measured by adding 90 µL quenched fluorescent substrate per well (total volume 100 µL/well, 10 µL/5mL enzyme buffer, 150 mM NaCl, and 50 mM Tri-HCl (pH 7.3), Fluorogenic Peptide Substrate, R&D systems, Cat#ES001 & ES011, Minneapolis, MI, USA). Fluorescence was monitored by a Fluoroskan Ascent™ (Thermo Scientific, Vantaa, Finland) Microplate Fluorometer (Cat#1506450, Thermo Scientific, Vantaa, Finland) (Cat#ES001 for Matrix Metalloprotease at an excitation of 320 nm, emission at 405 nm; Cat#ES011 for Kallikrein at an excitation of 390 nm, emission at 460 nm) over 400 mins or until activity had ceased. Data was collected using Ascent^® Software v2.6 (Thermo Scientific, Vantaa, Finland).