Cytation 3 cell imaging multi mode reader

SkBr3 cells and B-TEC (1 × 10⁵) were seeded in 24-well plates in regular growth medium until they reached a 70% to 80% confluence. The culture wells were then incubated in medium containing 2.5% charcoal-stripped FBS, treated and transferred into a time-lapse microscopy platform, equipped with a heated stage chamber (Cytation™3 Cell Imaging Multi-Mode Reader, Biotek, Winooski, VT). Cells were maintained at routine incubation settings (37°C, 5% CO₂) using temperature and gas controllers. To evaluate cell proliferation and motility, the images were recorded using Cytation 3 Cell Imaging Multimode Reader and the software Gen5 (BioTek, Winooski, VT) in 10 min intervals for 24 hours (cell proliferation) and 10 hours (cell motility). Then, the images were processed as a movie using the software Adobe Creative Cloud Premier Pro CC. Frames collected every 10 minutes are displayed at a rate of 10 frames s-¹.

Halicin was purchased from Carbosynth (Carbosynth Ltd., Compton, UK), while Mueller-Hinton broth was bought from Scharlab (Scharlab, S.L., Barcelona, Spain). Distilled water was generated through Milli Q (Millipore Corporation, Bedford, MA, USA) and had been used throughout this study. The measurement was made with CytationTM 3 Cell Imaging Multi-Mode Reader (BioTek Instruments, Winooski, VT, USA).

Cytodex^TM 3 (GE Healthcare, Chicago, IL, USA) microcarrier beads (0.5 g) were swollen in 50 mL DPBS and then autoclaved (121 °C, 20 min). HUVECs were coated on cytodex-3 beads at a density of 4 × 10⁵ cells/40 μL beads and incubated in suspension for 4 h with gentle mixing every 20 min. They were plated overnight on a T25 tissue culture flask. The next day, the suspension was transferred to a 15 mL conical tube and washed three times with 1 mL of M199 + penicillin/streptomycin to remove unattached cells. The cell-covered beads were re-suspended in a 2 mg/mL fibrinogen (Sigma-Aldrich St. Louis, MO, USA) solution containing 0.15 U/mL aprotinin (Sigma-Aldrich). Aliquots were mixed with thrombin (Sigma; 0.625 U/mL), distributed in 12-well plates (150 mL/well), and left to precipitate for 5–10 min. After further incubation (10 min, 37 °C, and 5% CO₂), a medium containing 25 ng/mL VEGF or 30 ng/mL bFGF and 33.57 μM of UA was added to cover the precipitate. Bead assays were monitored for 10 days. Images of beads were captured using a microplate Cytation^TM 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA) and analyzed using automatic WimSprout Image Analysis software (ibidi GmbH, Gräfelfing, Germany).

The effect of lichen metabolite UA on the viability of HUVECs was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromine (MTT) reduction assay. First, HUVECs were stimulated with different VEGF and bFGF concentrations (10–40 ng/mL) to determine the optimal dose of either factor to promote endothelial cell proliferation. Subsequently, cells were plated in gelatin-coated 96-well micro-culture plates (5 × 10³ cells/well) containing 80 μL of medium and incubated for 24 h at 37 °C in a 5% CO₂ atmosphere. Then 24 h after seeding, 20 μL of vehicle (0.1% DMSO) and aliquots of drug solutions (10, 50, 100 μM) in the presence or absence of VEGF (25 ng/mL) or bFGF (30 ng/mL) were added to HUVECs in triplicate wells. After 48 h of culturing, MTT reagent was added to each well at a final concentration of 0.5 mg/mL, and cells were incubated for another 4 h at 37 °C. Then 100 μL of sodium dodecyl sulfate (SDS) was added to each well, and the plate was kept on a shaker for 15 min to dissolve the formazan crystals formed in intact cells. A microplate Cytation^TM 3 Cell Imaging Multi-Mode Reader(Biotek, Winooski, VT, USA) was used to measure the absorbance at wavelength 595 nm. Results were expressed as the percentages of reduced MTT, assuming the absorbance of control cells as 100%.

The motility of HUVECs was assayed using a wound healing assay. Briefly, HUVEC cells were plated in a gelatin-coated 24-well plate at 1 × 10⁵ cells/well and incubated in the cM199 medium until a uniform monolayer was formed. A linear scratch was then made by using an SPL Scar^TM scratcher (SPL Life Science, Pocheon, Korea). The cells were washed three times with 1 mL of de-ionized phosphate-buffered saline (DPBS; pH 7.4) and treated with UA (33.57 μM) in the presence or absence of 25 ng/mL of VEGF and 30 ng/mL of bFGF in ECGF and heparin-free medium. At the end of the experiment, cells were fixed with methanol, stained with CellTrace^TM Yellow (Thermo Fisher, Rockford, IL, USA), and then captured under an inverted light microscope. The wounded area was photographed at the start (t = 0 h) and after 24 h. Images of the wounded areas were captured using a microplate Cytation^TM 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). The experiments were performed in duplicate wells and repeated three times with cells from different donors.

The day before performing the tube formation assay, a Matrigel matrix (BD Biosciences, Billerica, MA, USA) was incubated on ice overnight. On the day of the assay, 50 μL of Matrigel matrix was added to a 96-well plate and incubated at 37 °C, 5% CO₂ for 30 min. The cells with tested compound (UA 33.57 μM) and in the presence or absence of growth factors (VEGF 25 ng/mL; bFGF 30 ng/mL) were transferred to each well containing the Matrigel matrix. The plates were incubated for 3 to 16 h according to the size of the tube. Tube formation was quantified by measuring segment numbers, total segment length, number of junctions, and number of nodes in three random x 3 magnification fields per well, using a microplate Cytation^TM 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). The data analysis was performed using ImageJ software.

BrdU incorporation was used to analyze cell proliferation activity and was monitored by quantification of BrdU introduced to the genomic DNA during cell growth after EOT treatment (final dilutions ranged between 0.72–0.023 µg/mL and 0.72–0.0056 µg/mL, respectively). DNA synthesis was assessed using a colorimetric cell proliferation ELISA assay (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer’s protocol. The color intensity was measured with a Cytation^TM 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA at 450 nm (reference wavelength: 690 nm). The results were expressed as a fold of the control. All experiments were performed in triplicate. For the following analyses final dilutions (calculated from MTS and BrdU assays) of 0.12 µg/mL (MDA-MB-231 cells) or 0.13 µg/mL (MCF-7 cells) were used.