Ez cytox

Silica gel 60 powder and silica gel 60 F₂₅₄ aluminum sheets and glass plates for thin-layer chromatography (TLC) were obtained from Merck Supelco (Darmstadt, Hesse, Germany). Sephadex LH-20 (LH20_100) power was obtained from Millipore (Cytiva, Marlborough, MA, USA). High-pressure liquid chromatography (HPLC) was conducted using a Shimadzu LC-10 system (Tokyo, Japan). Breast cancer cell viability was determined using a cell viability assay kit (EZ-Cytox, DoGenBio, Seoul, Korea). The other chemicals and organic solvents were obtained from Sigma (St. Louis, MO, USA).

MTT assay was used to assess PDAC cell viability in response to various chemicals. Cells were seeded in 96 well plates (2500 cells/well), incubated for 24 h at 37 °C with 5% CO₂, and then treated with the required drug concentration for another 24 h before adding 10 μL of MTT reagent (EZ-CYTOX, DoGenBio Co., Ltd., EZ-3000, South Korea) and incubating for 90 min. The absorbance was taken at 450 nm using the Thermo Scientific Multiskan GO (Waltham, MA, USA). An assay was performed in triplicate.

Cells (1 × 104/well) were seeded in a 96-well plate. After incubation for 24 h, increasing concentrations of fluorouracil were added. Subsequently, 20 μL of water-soluble tetrazolium salt solution (Ez-Cytox; DoGenBio, Seoul, Korea) was added to each well, and the cells were incubated at 37 °C under 5% CO₂, for 3 h. Finally, the absorbance at 450 nm was measured using a microplate reader (E-MAX; Molecular Devices, San Jose, CA, USA).

Cells were plated (1 × 10³ cells/well) in 96-well plates and incubated for 18 h in a humidified incubator. After incubation, cells were transfected with control/IL13Rα2 siRNA or treated with DMSO (0.1%) control/the indicated treatment for 24, 48, or 72 h. After incubation, 20 µL of EZ-Cytox (DoGenBio, Republic of Korea) was added to the medium. After 4 h, absorbance was measured at 460 nm wavelength by a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

The effect of marine EVs on proliferation in normal or inflammatory environments was evaluated using the water-soluble tetrazolium salt (WST) assay (EZ-cytox, DoGenBio, Seoul, Korea). Briefly, based on the protein concentration measured by the Bradford assay, mEVs were diluted to 5, 10, 15, and 20 μg/mL in FBS-free RPMI-1640 medium and treated onto RAW 264.7 cells for 24 h. In the inflammatory environment, the same concentration of LPS (50 ng/mL) was used. The WST assay was performed according to the manufacturer’s instructions.
To check the cell viability, highest concentration marine EVs (20 μg/mL) in FBS-free medium were treated on RAW-264.7 for 24 h. Then, the medium was changed using the live and dead assay kit containing calcein-AM and ethidium homodimer (Invitrogen) in Hanks’ balanced salt solution (HBSS, Welgene, Gyeongsan, Korea) for 1 h. After 1 h, live and dead cells were observed using a fluorescence microscope (Carl Zeiss).