Alamar blue assay kit

PCS was purchased from Alsachim (Illkirch-Graffenstaden, France). NAC was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against NF-κB p65, p-ERK, p-JNK and p-P38 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against ALP were obtained from Abcam (Cambridge, UK). Anti-RUNX2 and other antibodies can be obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Alamar Blue assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

After transfection of shRNA, cells were treated with T₄ for 72 h. Cell viability was determined by using the Alamar Blue^® Assay Kit (ThermoFisher Scientific, DAL1100). At the time of detection, medium was removed, and cells were incubated with Alamar Blue^® reagent for 2 h at 37 °C according to the manufacturer’s instructions. Plates were then analyzed using a VersaMax Microplate reader (Molecular Devices, San Jose, CA, USA) at a wavelength of 570 nm, with 600 nm as a reference.

MCF-7, MDA-MB-231, and Vero cells were plated at a density of 4 × 10³ cells/well in 96-well plates. Cell viability was determined by using the Alamar Blue^® Assay Kit (Thermo Fisher Scientific, Watertown, MA, United States) at 72 h after treatment. Medium containing different drugs was replaced daily. At the time of detection, medium was removed, and cells were incubated with Alamar Blue^® reagent for 2 h at 37°C according to the manufacturer’s instructions. Plates were then analyzed using a VersaMax Microplate reader (Molecular Devices, San Jose, CA, United States) at a wavelength of 570 nm, with 600 nm as a reference.

AlamarBlue assay kit (DAL1025) was purchased from Thermo Fisher. Cells were cultured in Corning 96-well black plates with clear bottom and treated with drugs. Seventy-two hours later, AlamarBlue dye was added to each well according to the protocol and the plate was read by the POLARstar Omega Microplate Reader (BMG LABTECH) (excitation at 544 nm and emission at 590 nm). In the colony formation assay, cell lines were planted in six-well plates and treated with drugs for 14 days. Cell colonies were fixed by 100% methanol and then stained by 0.5% crystal violet in 25% methanol. Images captured by a scanner are representative of at least three independent experiments.

Gefitinib (#G-4408), erlotinib (#E-4007), trametinib (#T-8123), crizotinib (#C-7900) and paclitaxel (#P-9600) were purchased from LC Laboratories (Woburn, MA, USA). Cisplatin (#479306) was purchased from Sigma Aldrich (St. Louis, MO, USA). 2D and 3D cultures were incubated in the presence of the drugs for 72 hours. Cell viability was determined using the commercially available alamarBlue^® assay kit (Thermo Fisher Scientific, Madison, WI, USA). Apoptosis was analyzed using the Caspase-Glo^® 3/7 assay multiplexed with the MultiTox-Fluor GF-AFC life stain (Promega, Madison, WI, USA). Luminescence and fluorescence intensities were measured using the Paradigm detection platform (Molecular Devices, Sunnyvale, CA, USA). Statistical analysis was done using the GraphPad Prism Software 7.03 (GraphPad Software Inc., La Jolla, CA, USA). For the in situ analyses of apoptosis in cocultures the cells were incubated in the presence of 50 nM Gefitinib or 20 nM crizotinib for 24 hours and processed for immunofluorescent microscopy using the cleaved caspase-3 antibody (Cell Signaling Technology, #9661) that specifically detects apoptotic cells.

Polyethylene glycol (₂₀₀₀)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (ammonium salt) (PEG₂₀₀₀-DSPE), DSPE-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rh-DSPE), and N-maleimide-PEG₂₀₀₀-DSPE (ammonium salt) (Mal-PEG₂₀₀₀-DSPE) were purchased from Avanti Polar Lipids (CA, USA). The EDB-FN-specific peptide N'-CSSPIQGSWTWENGK(C)WTWGIIRLEQ-C' was a custom-synthesized peptide ordered from Anygen Corp. (Gwangju, Republic of Korea). The mouse anti-EDB-FN antibody and anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody were purchased from Abcam (Cambridge, UK). Docetaxel (DTX) and Sepharose CL-4B columns were purchased from Sigma-Aldrich (MO, USA), mounting solution was purchased from Dako Diagnostics (Glostrup, Denmark), and an Alamar Blue assay kit was purchased from Thermo Fisher Scientific (MA, USA). All reagents were laboratory grade and were used as received.