Cells were seeded into 96-well plates at a density of 1 × 104 cells/well. After 16–24 h, cells were treated with the extracts from petioles and roots of R. rhaponticum and R. rhabarbarum and stilbenes (RHPG and RHPT), at concentrations of 1–100 µg/mL, for 24 h. After incubation, the cell culture medium was removed, and wells were rinsed twice with 0.02 M phosphate-buffered saline (PBS) containing Ca2+/Mg2+ (0.8 mM/0.4 mM), incubated in PBS containing Ca2+/Mg2+, 5.5 mM glucose, and 0.0125 mg/mL resazurin. HUVECs viability was estimated by measurements of the ability of live cells to reduce non-fluorescent resazurin to resorufin, a fluorescent product. After a 3 h incubation, resorufin fluorescence was measured (λex = 530 nm, λem = 590 nm), using the Fluoroscan Ascent microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) [20 (link)]. The metabolic activity of control HUVECs (untreated with the examined extracts and stilbenes) was assumed as 100% of cell viability. Samples treated with 1% Triton-X100 were reference samples, with no viable cells (0% of viability). In cell samples treated with the examined extracts or stilbenes, a decrease in cell viability ≥ 20% (compared to control/untreated HUVECs) was assumed as a cytotoxic effect.
Fluoroscan ascent microplate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Fluoroskan Ascent microplate reader is a compact and versatile instrument designed for fluorescence, luminescence, and absorbance measurements. It features a high-sensitivity optical system and advanced data analysis capabilities to support a variety of applications in life science research and diagnostics.
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5 protocols using fluoroscan ascent microplate reader
1
Cytotoxicity Evaluation of Rhubarb Extracts
2
Cytotoxicity Assay of PAD Inhibitors
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Cytotoxicity was analyzed by resazurin reduction to fluorescent resorufin, as described previously [24] . Cells were seeded onto 96-well plates at a density of 5, 000 and 7, 000 cells/well for HUVECs and HMEC-1 cells, respectively. After 16-20 h, the cells were treated with PAD inhibitors at a concentration range of 0.1-10 μm for 24 h. After the indicated time, the culture medium was removed, and cells were washed with phosphate buffered saline (PBS) containing Ca 2+ /Mg 2+ and 5.5 mM glucose. The cells were then incubated for 2 h in the washing buffer supplemented with 0.0125 mg/ml resazurin. After incubation, fluorescence was read on a Fluoroscan Ascent microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at λ ex = 530 nm and λ em = 590 nm.
3
Quantifying MMP Activity in HMEC-1 Cells
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The total activity of MMPs in HMEC-1 cells treated with PAD inhibitors was measured using a fluorometric assay (MMP Activity Assay Kit, ab112146; Abcam, Cambridge, UK) according to the manufacturer's instructions. Briefly, HMEC-1 cells were seeded onto 6-well plates at a density of 700, 000 cells/well, followed by treatment with the highest concentration of all inhibitors. As a positive control for MMP inhibition, 20 µg/ml doxycycline (DOX) was used. After 16 h, the supernatant was collected and incubated for 3 h with 2 mM 4-aminophenylmercuric acetate (APMA) to activate the MMPs. After that, the supernatants activated with APMA were transferred to a U-bottom 96-well plate, and MMP Green Substrate was added to the samples. Subsequently, fluorescence was read in all samples at 5-minute intervals using a Fluoroscan Ascent microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at λ ex = 490 nm and λ em = 525 nm.
4
Cytotoxicity Evaluation of Polypeptide Particles
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ARPE-19, HEK-293 and BEAS-2B were used for cytotoxicity evaluation. 104 cells per well were cultured in a 96-well plate (200 μL/well) in DMEM-F12 containing 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin (72 h, 37 °C, a humidified atmosphere of 5% CO2). After that, the cells were cultivated in the culture medium containing test empty polypeptide particles at the concentrations from 3 to 125 μg/mL for 24 and 72 h. The viability was evaluated using MTT-assay. For that, culture medium was aspirated and 100 μL/well of MTT solution (1.0 mg/mL in DMEM-F12) was added. The plate was incubated for 2 h. Finally, the solution was removed and 100 μL of DMSO was added to each well. After 10 min of gentle shaking, the solution absorbance was measured at 570 nm with the use of Fluoroscan Ascent microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). The relative cell viability (%) was calculated as following:
5
Resazurin-based Cell Proliferation Assay
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Cell proliferation was analyzed using the ability of live cells to reduce resazurin to fluorescent resorufin, as it was described previously [50 (link)]. Both endothelial cell types were seeded onto 96-well plates at a density of 5000 and 7000, respectively for 24 h and 72 h incubation. After 16–20 h the cells were treated with EGCG (Sigma-Aldrich, Saint Louis, Missouri, USA) at the concentration range of 5–200 μM for 24 h or 72 h with a medium change every 24 h. After the time indicated above the culture medium was removed, cells were washed with PBS containing Ca2+/Mg2+ and 5.5 mM glucose. Then the cells were incubated for 2 h in PBS containing Ca2+/Mg2+, 5.5 mM glucose with 0.0125 mg/mL resazurin. After incubation fluorescence was read on a Fluoroscan Ascent microplate reader (Thermo-Fisher Scientific, Waltham, MA, USA) at λex = 530 nm, λem = 590 nm (Table 1 ).
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