Celltiter 96 aqueous one solution cell proliferation reagent

Cells were plated on 96-well plates and treated with various concentrations of each drug alone or in combination, at a constant ratio. Following daily treatments for 4 days, the cells were assayed with CellTiter 96 Aqueous One Solution Cell Proliferation Reagent (Promega, Madison, Wi, USA). The absorbance values were used to determine the fraction of cells affected by each treatment and to determine the combination indices (CIs) according to the Chou–Talalay method, using CompuSyn software: CI <1 indicates synergism, CI = 1 indicates an additive effect, and CI >1 indicates an antagonistic effect.

In vitro RaST experiments were performed in 24 well plates containing 4 x 10⁵ cells per well in 1 mL of CM. The treatment was initiated by the addition of either VLA-4-TC-NM (5 µL per well, 0.5 µg TC) or αvß3-TC-NM (5 µL per well, 0.5 µg TC), or their combination. The NM were allowed to bind to target receptors at 4 °C for 1 h. The medium with unbound NM was carefully removed and replaced with 1 mL fresh medium. At this time, 3.7 MBq/mL (0.1 mCi) [¹⁸F]FDG in saline was added to the appropriate wells, and the plates were placed in the 37 °C/5% CO₂ humidified atmosphere for the indicated amounts of time. After the completion of treatment, the medium was carefully removed and 0.5 mL of PBS containing 50 µL MTS reagent (CellTiter 96^® Aqueous One Solution Cell Proliferation Reagent, Promega, Madison, WI) was added to each well to determine the percentage of live cells. The plates were incubated at 37 °C for 1-4 h to allow the tetrazolium compound to be reduced to form formazan dye. The resulting absorbance was detected at 490 nm with a Synergy/NEO2 multi-mode reader (BioTek, Winooski, VT). The data were reported as a percent of live cells compared to the untreated cells (100%).

Different G. neuberthii extracts were evaluated on cell proliferation by the MTS metabolic viability assay, according to Guamán-Ortiz et al. (2015). [10 (link)] Briefly, 2 × 10³ cells were seeded in 96-well plates in 100 μL of medium per well and incubated for 24 h. Then, cells were treated in triplicate for 48 h either with 50 μg/mL of each extract or 0.3 μg/mL Dxo. Four hours before finishing the treatment, 20 μL of Cell Titer 96 Aqueous One Solution cell proliferation reagent (Promega, USA) was added to each well. The plates were then maintained for 4 h at 37°C; the absorbance of each sample was measured with a microplate reader (Megallan, Tecan, Switzerland) at a wavelength of 492 nm. Absorbance from control was used as reference values (100% of viability) to normalize the data of treated samples. In order to calculate the IC₅₀, the most active extracts were selected and exposed to the cells for 48 h with increasing concentrations (5–50 μg/mL) and processed as above.

Human TF1 cells (Sigma-Alrich, St. Louis, MO, USA) were cultured for 1 week in RPMI-1640 medium containing 10% FBS with 2 ng/mL rhGM-CSF (R&D System, Minneapolis, MN, USA). Cells were then washed and resuspended in RPMI-1640 + 10% FBS to a concentration of 420,000 cells/mL and replated on 96-well microplates (12,600 cells per well in 30 mL). The medium was removed 1 h after TF1 cells replating, and to the wells was respectively added 100 μL of RPMI-1640 containing TG pig-derived saliva or purified hNGF. After incubation for 40 h at 37 °C and 5% CO₂, the medium was changed with 50 μL/well RPMI-1640 containing 10% FBS. To each well was then added 20 μL of “Cell Titer 96 Aqueous One Solution Cell Proliferation” reagent (Promega, Madison, WI, USA). The plate was incubated at 37 °C for 3 h in a humidified, 5% CO₂ atmosphere. The absorbance of each well at 490 nm was recorded using a microplate reader (model: Synergy-H1, Bio Tek, Shoreline, WA, USA) after 3 h of incubation.

Cell lines were seeded in a 96-well plate (5 × 10³ cells/well) and cultured for 24 h in 100 μL of culture medium. For transfection, 15 μl of Opti-MEM (Thermo Fisher Scientific), containing 7.5 pmol of siRNA and 0.15 μL of Lipofectamine 2000 or Lipofectamine RNAiMAX (Thermo Fisher Scientific), was added to the cells in each well and cultured for 24, 48, or 72 h. The viable cell count was measured by CellTiter 96 AQueous One Solution Cell Proliferation Reagent (Promega K.K., Tokyo, Japan). Several human HEG1 siRNAs designed by Enhanced siDirect were used; HEG1 siRNA mix 1, a mixture of H1097 and H2674 (1:1); H3059, 5′-GCGAAUGCGUCGCAGACAACA-3′ and 5′-UUGUCUGCGACGCAUUCGCCA-3′; and H9106, 5′-CUGGCGUUCUAGUCAGUAAAA-3′ and 5′-UUACUGACUAGAACGCCAGAC-3′. Four commercially available siRNAs against human HEG1 were also tested: HEG1 siRNA mix 2, sc-78365 (Santa Cruz Biotechnology); S3816, SASI_Hs02_00353816; S3817, SASI_Hs02_00353817; and S3818, SASI_Hs02_00353818 (Sigma-Aldrich Japan K.K.). As a negative control, MISSION siRNA Universal Negative Control (SIC-001) was used. HiLyte Fluor 488-labeled universal negative control siRNA was obtained from Nippon Gene Co (Tokyo, Japan). In tested cell lines, sufficient HiLyte Fluor-labeled siRNA was transfected with the lipofection reagent.

Ferroptosis inhibitor 3-amino-4-(cyclohexylamino)-benzoic acid, ethyl ester (FST-1, Cayman Chemical, Ann Arbor, MI) was used to investigate the involvement of Ferroptosis in RaST-mediated cell death. MM1.S were plated and either left untreated or treated with VLA-4-TC-NM RaST, as well as VLA-4 and FDG controls, as described in the in vitro RaST section. FST-1 (10 µM) was added concomitantly with the VLA-4-TC-NM RaST treatment. After 72 h incubation at 37 °C/5% CO₂ humidified atmosphere, the CM was carefully removed from each well and replaced with 0.5 mL of PBS containing 50 µL tetrazolium compound (CellTiter 96® Aqueous One Solution Cell Proliferation Reagent, Promega, Madison, WI) to determine the percentage of live cells. The plates were incubated at 37 °C for 1-4 h to allow the tetrazolium compound to develop. The resulting absorbance was detected at 490 nm with a Synergy/NEO2 multi-mode reader (BioTek, Winooski, VT).