Xmark spectrophotometer

For the analysis of drug-resistance, cells (1 × 10⁵ cells/well) were cultured in 12-well cell culture dishes and were cultured overnight at 5% CO₂ and 37 °C. Afterward, cells were incubated with various doses of doxorubicin (DOX; 0.01, 0.1, 0.25, 0.5, and 1 µM, respectively) and were cultured for another 48 h. After 48 h incubation, cells were counted or medium was exchanged with a fresh medium containing EZ-Cytox (Daeil Lab Service, Seoul, Korea) and was incubated for an additional 3–4 h at 37 °C in an atmosphere of 5% CO₂. The absorbance was measured at 450 nm by using a microplate reader Bio-Rad x-Mark™ spectrophotometer (Bio-Rad). The results were represented as a percentage (%) of surviving cells, as described previously [46 (link),98 (link)].

Two mammalian cell lines, including human embryonic kidney cells (HEK293T) and human breast cancer cells (MCF7) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone^TM, Logan, UT, United States) supplemented with 10% FBS (Hyclone^TM) and 1% penicillin/streptomycin (Hyclone^TM) and incubated at 37°C and 5% CO₂ up to 80% confluence. Cellular adherence to the substratum was disrupted using Accutase (Innovative Cell Technologies, San Diego, CA, United States). In total, 1 × 10⁴ to 4 × 10⁴ cells in each well of a 96-well plate containing 8, 16, 32, and 64 μg/mL of ModoCath peptides were incubated for 24 h at 37°C and 5% CO₂. Additionally, HEK293T cells were incubated in the FBS-free medium. Triton X 100 (Sigma Aldrich) was used as a positive control for complete cell lysis, and untreated cells were used as the negative control. After incubation, the medium was removed from the wells, and 10 μL of coloring solution (Cell Proliferation Reagent WST-1^TM; Sigma Aldrich) and 100 μL of DMEM (Hyclone^TM) were added to the wells in accordance with the manufacturer’s protocol. Absorbance was measured for each well at 440 nm (peptide-treated and control) and 650 nm (background) and recorded as the OD, using a microplate reader (xMark^TM spectrophotometer; Bio-Rad). Cell viability was calculated using the following equation:
All experiments were performed in triplicate.

B16F10 cells were cultured at a density of 4 × 10⁴ cells/well onto 12-well plates for 24 h. Subsequently, each well was treated with 200 nM of α-MSH and OOM-SC-EVs (1.5, 3, 9, and 15 × 10⁸ particles/mL) or arbutin (100 μM) and then incubated for 48 h. Each well was washed with PBS, and the cells were harvested with PBS and suspended in 50 mM of phosphate buffer (pH 6.8) containing 1% Triton X-100. After vortexing, the mixture was incubated at 80 °C for 30 min and then subjected to thawing at room temperature. After centrifugation at 1000× g for 10 min, 40 μL of the supernatant and 100 μL of 10 mM L-DOPA (Sigma, 333786) were added to a 96-well plate and incubated at 37 °C for 1 h. Absorbance was then measured at 405 nm using a Bio-RAD x-MarkTM spectrophotometer. Each absorbance value was normalized to that of total protein.

B16F10 cells were seeded onto 12-well plates at a density of 4 × 10⁴ cells/well and cultured with RPMI medium containing 10% FBS overnight. Subsequently, 200 nM α-melanocyte-stimulating hormone (α-MSH) (sigma, M4135) and dose-dependent exposure of OOM-SC-EVs (1.5, 3, 9, and 15 × 10⁸ particles/mL) or arbutin (100 μM) (Sigma, A4256) were administered and incubated for 60 h. Further, to measure extracellular melanin, we transferred 100 µL of culture medium to a new 96-well plate, and absorbance was detected at 405 nm wavelength with a Bio-RAD x-MarkTM spectrophotometer. Additionally, for intracellular melanin measurement, each well was washed with PBS and then dissolved with 200 µL of 1 N NaOH for 1 h at 80 °C. Absorbance was then measured at 405 nm. The extracellular and intracellular melanin levels were normalized to the total protein concentration.

HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone^TM, Logan, UT, USA) with 10% fetal bovine serum (FBS; Hyclone^TM) and 1% penicillin/streptomycin (Pen-Strep; Hyclone^TM) up to 80% confluence at 37 ℃ and 5% CO₂. Cells were detached from the plate by adding 0.25% trypsin-EDTA solution (Gibco^TM; Carlsbad, CA, USA). In a 96-well plate, 2 × 10⁴ cells were seeded per well and cultured for 24 h. Subsequently, the medium was replaced with 100 μL DMEM (Hyclone^TM) containing 10% heat-inactivated FBS. Peptides were added to each well at concentrations of 64 μg/mL and 160 μg/mL. As references for complete cell lysis, 64 μg/mL melittin (Sigma-Aldrich) and Triton X-100 (Sigma-Aldrich) were used. Untreated cells were used as negative controls. After 24 h incubation at 37 ℃ and 5 % CO₂, 10 μL of coloring solution (Cell Proliferation Reagent WST-1^TM; Sigma Aldrich) was added to each well according to the manufacturer’s instructions. The absorbance at 450 nm (treated group and control) and 650 nm (background) was measured in each well using a microplate reader (xMarkTM spectrophotometer; Bio-Rad). The cell viability was calculated using the following equation. All experiments were performed in triplicate.

The capacity of bee pollen hydroethanolic extracts to scavenge free radicals, namely DPPH^●, ^●NO and O₂^●−, was performed spectrophotometrically through in vitro microassays using 96-well plates. Microplate reader Bio-Rad Xmark spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) was used to measure the absorbances. The results were expressed as 25% and 50% maximal inhibitory concentration (IC₂₅ and IC₅₀, respectively) values (µg/mL). Each experiment was performed in triplicate, and seven different concentrations were tested.

We tested the antibacterial activity of the extracts in a dilution series (25–0.39 mg/mL) in triplicate using a micro-broth dilution test against P. alvei CCM 2051 provided by the Czech Collection of Microorganisms (Masaryk University, Brno, Czech Republic) as previously described [74 (link)] with modifications. Briefly, extracts were diluted 1:1 with Mueller–Hinton broth, yeast extract, potassium phosphate, glucose, and pyruvate (MYPGP), and 100 µL of the solution was pipetted to the first row of the microdilution 96-well plate. A two-fold dilution series was prepared by transferring the extract solutions (50 µL) to the next rows containing 50 µL of the MYPGP. Dilutions of methanol were included as a solvent growth control. The lyophilized bacteria were cultivated at 37 °C for ~24 or ~48 h on MYPGP agar. The inoculum was diluted in sterile water and adjusted to 0.5 McFarland units using the DEN-1 McFarland densitometer (BioSan, Riga, Latvia). The bacterial suspension was diluted 1:150 with the MYPGP (~10⁶ CFU/mL), and 50 µL was added to each well within 15 min. Bacterial growth was measured at 625 nm using an xMark spectrophotometer (Bio-Rad, Hercules, CA, USA). Minimum inhibitory concentrations (MICs) were defined as the lowest concentration of extract or standard that inhibited growth by at least 80% relative to the control (bacterial suspension with no extract/solvent).