Wallac1420 victor microplate reader

Luciferase activity was measured using the Steady-Glo® Luciferase Assay System (Promega, USA). Briefly, PC3-NF-κB-Luc cells were plated in 96-well plates at a density of 5×10⁴ cells/well. After 24 h, the cells were left untreated or treated with TNF-α (20 ng/ml) for 30 min and then lycorine (0 μM, 1 μM, 5 μM, 10 μM, 20 μM) and TPCA-1 (2 μM) were added for further 12 h. At the end of treatment, an equal volume of Steady-Glo Reagent was added into the culture medium in the microplate, and luciferase activity was measured using a Wallac1420 VICTOR microplate reader (Perkin-Elmer, Wellesley, MA, USA).
Cell viability was examined using the MTT method. Briefly, cells were plated in 96-well plates at a density of 5×10⁴ cells/well for 24 h and then treated with lycorine (0 μM, 1 μM, 5 μM, 10 μM, 20 μM). At the end of each treatment, 20 μL of MTT solutions (5 mg/ml) was added into each well and incubated for 2 h at 37 °C. After the medium was removed, 100 μL DMSO was added to each well to dissolve water-insoluble formazan crystals. The optical density of each well was measured with a Wallac1420 VICTOR microplate reader at 490 nm (Perkin-Elmer, Wellesley, MA, USA).

The 5TGM1 MM PC line was cultured with either Grem1-overexpressing or empty vector OP9 stromal cells for 72 h in a 24-well plate, as described above. Following incubation, cells were collected and lysed in 1 × Passive Cell Lysis Buffer (Promega). The 20 µL of cell lysate was transferred to a 96-well plate. Immediately prior to reading the plate, 100 µL of luciferase reaction buffer (5 mM MgCl₂, 30 mM HEPES, 150 μM ATP, 500 µg/mL of Coenzyme A, and 150 μg/mL D-luciferin) was added to the cell lysate. Luminescence was measured using a Wallac 1420 Victor Microplate reader (Perkin Elmer), with luminous intensity used as a direct measure of MM PC number.

To simultaneously allow quantitative and microscopic assessment of cell viability an (Sigma-Aldrich) staining was conducted. FDA freely passes the cell membrane being intracellularly hydrolyzed by nonspecific esterases generating the green-fluorescent fluorescein, whose fluorescence intensity is correlating with the cell viability [43 (link),77 (link),78 (link)]. In 96-well culture plates, supernatants were discarded and cells washed in PBS. An amount of 100 µL of 10 µg/mL FDA staining solution were added to each well and incubated for 5 min at 37 °C and 5% CO₂. To determine cell morphology and growth patterns, the wells were visualized using an Olympus IX-81 inverted fluorescence microscope (Olympus, Hamburg, Germany). For simultaneous quantification of cell viability, stained cells were washed in PBS and afterwards lysed in 150 µL 0.5% Triton X-100 (Sigma-Aldrich) for 5 min. Lastly fluorescence intensity was determined at a wavelength of 535 nm using a Wallac 1420 Victor microplate reader (Perkin Elmer, Waltham, MA, USA).
Proliferation was analyzed using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies) as described in the manufacturer’s instructions. The fluorochrome PicoGreen selectively binds double-stranded DNA (dsDNA) and thus permits the quantification of mononuclear cells such as MSCs.