Cell bind 96 well flat clear bottom black polystyrene microplates

The effect of the N-acyl amides on calcium mobilization was assessed using a high-throughput 96-wells plate assay adapted from our previous work (Rimmerman et al., 2008 (link)). TRPV1 (human), TRPV2 (mouse), TPRV3 (mouse), or TRPV4 (rat)-transfected HEK-293 (TRPV2 cells were a kind gift from Vincenzo DiMarco and the TRPV3 and 4 cells were a kind gift from Michael Caterina) cells were seeded on Cell BIND 96-well flat clear bottom black polystyrene microplates (Corning Life Sciences, Acton, MA) coated with 50 μg/mL poly-D-Lysine (PDL) under the cell culture hood at room temperature, allowing cells to attach to plates. Cells were grown to 75–85% confluence in a monolayer in the culture medium described above for 24–48 h. Prior to all experiments, cells were examined under the microscope to check for optimal confluency and normal morphology.

The fluorescent dyes (Hoechst 33342, HCS LipidTOX Green neutral lipids (H34475), HCS LipidTOX Red phospholipidosis (H34351), MitoTracker orange CMTMRos (M7510), carboxy-H2DCFDA (C400), and TOTO-3 Iodide (T3604)) were from Life Technologies, Invitrogen. The CellBIND 96 Well Flat Clear Bottom Black Polystyrene Microplates were purchased from Corning.