Multiscan ascent elisa reader

Viability of H69 and SW780 cells treated with calcium with or without electroporation was assessed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(carboxymethophenyl)-2-(4-sulfonyl)-2H-tetrazolium) assay (Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay; Promega). MTS assay is a colorimetric method that estimates the rate of metabolism in viable cells. After treatment 100 μl cell suspension (2.4 × 10⁵ cells/ml) was seeded in 96-well plates. After 24 hours incubation 20 μl MTS/PMS reagent was added to each well (final concentrations of 333 μg/ml MTS and 25 μM PMS). After 1 hour of incubation at 37°C and 5% CO₂ cell viability was assessed by measuring optical density (OD) using Multiscan-Ascent ELISA reader (ThermoLabsystems, Finland) at 490 nm with background measurements at 690 nm.

Intracellular calcium level was measured for the two cell lines, undifferentiated and differentiated. Cells were divided into seven groups: Calcium electroporation (0.5 and 1 mM) collected 15 and 60 min after treatment, calcium alone (0.5 and 1 mM) collected after 60 min, and untreated. Cells were electroporated in suspension using 1000 V/cm. After treatment, cells were suspended in 50 μl lysis buffer for 30 min, centrifuged, and the supernatant was collected. Total intracellular calcium concentration was measured using Calcium Colorimetric Assay Kit (Bio Vision) in 96-well plates. A standard curve was generated using serial dilutions of CaCO₃ (0–2 mg/dl). Absorbance was measured using Multiscan-Ascent ELISA reader (ThermoLabsystems) at 575 nm. The obtained results were expressed as Ca²⁺ level in mM where 1 mg/dl CaCO₃ corresponds with 0.1 mM Ca²⁺.

Cells (270 μl of 6.1 × 10⁶ cells/ml) suspended in HEPES buffer (10 mM HEPES (Lonza), 250 mM sucrose, and 1 mM MgCl₂ in sterile water) and 30 μl of CaCl₂ (0.5, 1, or 5 mM in final concentration) or HEPES for controls incubated 5 min at 37μ C in a 4 mm cuvette (Molecular BioProducts, Inc.). Cells were exposed to 8 pulses of 99 μs, 1 Hz, and 600, 800, or 1000 V/cm using a square wave electroporator (BTX T820, Genetronics, USA). After 20 min incubation at 37μ C, cells were suspended in culture medium and seeded in 96-well plates (3.1 × 10⁴ cells per 100 μl) and incubated for 24 h at 37μ C. MTS assay was performed using Multiscan-Ascent ELISA reader (ThermoLabsystems).