Celltiter 96 aqueous assay reagent

MTS tetrazolium cell viability assays were done according to the manufacturer's instructions (CellTiter 96 AQueous Assay reagent; Promega). Briefly, the CellTiter 96 AQueous One Solution Reagent was added to each well and incubated at 37°C for 3 hr. Cell proliferation was determined by measuring the absorbance at 490 nm using a microplate reader (Molecular Devices). The half-maximal inhibitory concentration (IC50) was obtained from the MTS viability curves using GraphPad Prism 5. Experiments were carried out in triplicate.

The cytotoxicity of the complexes was evaluated using MTS-based CellTiter 96 AQueous Assay reagent (Promega, Madison, WI, USA) according to manufacturer’s instructions. Briefly hASCs were plated at a density 1 × 10⁴ cells per well in flat-bottomed 96-well plates (Corning Costar, Cambridge, MA, USA) and were incubated at 37 °C. After 24 h, the hASCs were incubated with 4 µL of pDNA/PEI binary complex or 6 µL of pDNA/PEI/HA ternary complexes for 24 or 48 h. For the MTS assay, 10 µL of MTS reagent was added into each well containing 100 µL of culture medium, and then the plates were incubated for 3 h at 37 °C. Absorbance of the wells was detected at 490 nm by a microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA).

MTS cell survival was measured using the CellTiter 96 Aqueous Assay reagent (Promega, WI, USA). LDH Release was measured using the CytoTox 96 Non-Radioactive reagent (Promega). Plates were recorded at 490 nm and 690 nm (plate background). Cell survival was measured using Red Neutral staining as described previously [59 (link)]. Plates were recorded at 540 nm. Apoptosis was measured with the RealTime-Glo Annexin V Apoptosis Assay (Promega) or with AnnexinV-FITC and propidium iodide (PI) (BioLegend) before flow cytometry analysis (FACSCalibur, BD, NJ, USA) driven by BD CellQuest software (BD). All assays were performed according to manufacturers' instructions.

RRM2 and scrambled control siRNA were purchased from Santa Cruz Biotechnology Inc. Cells were seeded at 2 × 10⁵ cells per well in 6-well plates filled with 2 mL antibiotic-free normal growth medium supplemented with FBS and then incubated at 37°C in a 5% CO₂ incubator for 24 hours. A total of 4 μL of 10 nmol/mL RRM2 or control siRNA was transfected into MDA-MB-231 or ZR-75-1 cells by using the transfection RNAiMAX reagent (Invitrogen, Carlsbad, CA). Cells were incubated in the transfection medium for 6 hours and then placed in normal cell culture medium. The inhibition of RRM2 and RRM2B was measured by using Western blot analysis.
For the invasion assay
[22 (link)], 2.5 × 10⁴ cells were seeded on the Matrigel™ (BD company) insert of the 24-well chamber. After incubation for 72 hours in 5% CO₂ at 37°C, the top of the Matrigel™ inserts were wiped with a cotton-tipped swab to remove cells that had not migrated through the membrane. The cells on the lower surface of the membrane were stained with 0.5% Coomassie blue (dissolved in 50% ethanol) and counted. Each experiment was performed three times.
Cytotoxicity was assessed using the MTS assay, according to the manufacturer’s instructions (CellTiter 96 AQueous Assay reagent; Promega) on 10 replicates of 2,500 cells per well in a 96-well plate treated with test drugs for 72 hours.