A TRPV1 expressing clone of the human SH-SY5Y neuroblastoma cell line (Figure 1) was obtained by stable transfection, using puromycin-containing selection medium. Prior to Ca2+ measurements the TRPV1-SH-SY5Y cells were cultured in 96-well plates to confluency. Acute increase in the intracellular free Ca2+ level was measured in a semi-HTS fluorescence reader (FlexStation II, Molecular Devices) using Fura-2/AM. The ratio of fluorescence at 340 (Ca2+-bound Fura-2)/380 (Fura-2) nm excitatory wavelengths was registered without interruption before and during the 2 min exposure to the test compounds. The mean value (% increase of basal Ca2+ level) from triplicate wells in the 96-well plate was monitored for each concentration from each experiment. The TRPV1 antagonist capsazepine was added simultaneously with each concentration of the chemicals in three sister wells to confirm TRPV1-mediated Ca2+ influx. The intracellular Ca2+ increase induced by the specific TRPV1-agonist capsaicin was set to 100% response for each experiment and the effect of the test products was calculated as percent of the capsaicin induced response. All test compounds were diluted in HKR-buffer and the addition to the cells was performed robotically during measurements by the FlexStation II reader.