Synergy 4 hybrid microplate reader

The HeLa and MCF-7 cancer cells were seeded in a 96-well plate with the density of 10,000 cells per-well (total medium volume of 100 μL). 24 hours post seeding, the solutions with a serial of concentrations of doxorubicin-peptide mixture or free doxorubicin in 100 μL of medium were added to each well (five wells for each concentration). Cells without the treatment of the compounds were used as the control. The MTT assays were performed after an extra culture time of 24 hours. All compounds were removed and 90 μL of fresh medium was added for each well, 10 μL of MTT solution (5 mg/mL) was added and incubated for 4 hours in 37°C. Pipette out the spent media, formazon crystals at the bottom of each well were dissolved in 100 μL DMSO. After 15 minutes shaking at room temperature, absorbance at wavelength of 490 nm was tested using a BioTek Synergy™ 4 Hybrid Microplate Reader. The experiment was repeated for 3 times. IC₅₀ values for the inhibition of cell viability were calculated from pharmacological inhibitory response curves using software Prism 5.0.

The synthesized compounds were characterized using ¹H NMR (Bruker ARX 400). LC-MS spectrometric analyses were performed at the LCMS-20AD (Shimadzu) system. HPLC was conducted at LUMTECH HPLC (Germany) system using a C18 RP column with MeOH (0.05% of TFA) and water (0.05% of TFA) as the eluents. Rheology was performed on an AR 2000ex (TA instrument) system using a parallel plates (40 mm) at the gap of 500 μm. MTT data was recorded on a BioTek Synergy™ 4 Hybrid Microplate Reader. TEM images were done on a Tecnai G2 F20 system, operating at 200 kV. The zeta potentials of the self-assembled nanofibers were measured by a zeta potential analyzer (Zeta Pals, Brookhaven Instruments, Huntsville, NY, USA). Before measurements, the hydrogels formed by adding different equiv. of doxorubicin to the 0.5 wt% peptide solution and peptide solution itself were all diluted for 5 times by 1 × PBS, affording corresponding nanofiber solutions. The zeta potential was then measured with palladium electrodes at 25°C, and the mean value of three readings was taken.

Cells were seeded onto a black 96-well plate (BrandTech Scientific, CAT# 91-425TB) at 25,000 cells per well in 100 µL of medium. The following day, the plate was exposed in triplicate to increasing doses of TNFα (50 pg/mL to 5 ng/mL), butyrate (50 µM to 7.5 mM), as well as co-treatment of 500 µM butyrate and TNFα (50 pg/mL–5 ng/mL) by adding 100 µL of 2× chemical stocks. Next, 50 µM menadione was added 4 h prior to the 24 h time period and served as a positive control for ROS production. After 24 h of incubation, 100 µL was removed. A 40 µM H2DCFDA dye solution (2×) was prepared and vortexed. Next, 100 µL of this mixture was added to each well using a multichannel pipette. The plate was mixed at 400 rpm via orbital shaker; then, it was incubated in 5% CO₂ at 37 °C for 45 min. Fluorescence was measured at an ex/em of 485/535 nm on a BioTek Synergy™ 4 Hybrid Microplate Reader using the autogain feature. The data represent the combination of two technical replicates normalized to the medium control (n = 3 wells/technical replicate for experimental conditions).