Infinite 200 pro series microplate reader

Calcium mobilization was performed as described previously [30 (link)]. The SKOV3 cells were harvested with Cell Stripper (Mediatech, Herndon, VA, USA), washed twice with PBS and resuspended to 5 × 106 cells/ml in Hank’s balanced salt solution(140 mM NaCl, 5 mM KCI, 10 mM HEPES, pH7.4, 1 mM CaCI₂, 1 mM MgCl₂, 1 mg/ml glucose) containing 0.025% BSA. Subsequently, the cells were loaded with 3 μM Fura-2 acetoxymethyl ester derivative (Fura-2/AM) (Molecular Probes, Eugene, OR, USA) for 30 min at 37°C. The cells were washed once in Hank’s solution, and then resuspended in Hank’s at a concentration of 3 × 10⁷ cells/ml. These cells were subsequently stimulated with 100 nM PAF. The calcium flux was measured using excitation at 340 and 380 nm in a Tecan Infinite 200 pro series Microplate Reader (Tecan, Switzerland). When required, the cells were treated with WEB2086 for 1 hour in serum-free McCoy’s 5A medium prior to the experiment.

Antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA) using goat anti-human IgG F(c) as well as goat anti-human kappa chain peroxidase conjugated antibody (Rockland, USA) and SeramunBlau^® (Seramun, Germany) as substrate. Absorption at 450 nm was measured by Infinite^® 200 PRO series microplate reader (Tecan, Switzerland). Amino acids and dipeptides were analyzed simultaneously by LC–MS after dansyl chloride derivatization as described below.

HEK293 cells were grown in regular DMEM supplemented with 10% FBS (Hyclone, GE Healthcare Life Sciences) and antibiotics at 37 °C, 5% CO₂. For transfection, rapidly growing cells were trypsinized and re-suspended in DMEM containing 10% FBS lacking antibiotics at a 0.1 × 10⁶ cells/ml concentration. We next added 50 μl of transfection reagent mixture (0.5 μl/well Lipofectamine 2000 in Opti-MEM; Thermo Fisher) to wells containing pre-spotted plasmids. We incubated the wells at room temperature for 20 min and subsequently added 100 μl of cells (0.1 × 10⁵ cells/well). Approximately 6 h after transfection, we replaced this medium with 150 μl of pre-warmed fresh DMEM containing 10% FBS and antibiotics and allowed the cells to grow for an additional 24–30 h. 36 h post-transfection, we replaced this medium with 150 μl of HEPES-buffered explant medium supplemented with luciferin (1 μM) and B-27 supplements; the plates were sealed with an optically clear film. We next loaded these plates into a 36 °C incubator and recorded bioluminescence expression with an Infinite^® 200 PRO series microplate reader (Tecan, Thermo Fisher).