Flexstation 3 plate reader

CHO-APJ cells expressing Nb5 or Nb17 were plated onto a 96-well plate at 80,000 cells/well and incubated at 37 °C overnight in 5% CO₂. On the day of the cAMP assay, cells were treated with IBMX at a final concentration of 750 µM for 20 min at room temperature. Cells then were treated with forskolin (20 µM), together with apelin-13 at various concentrations for 10 min. After these reactions, cells were lysed and their intracellular cAMP levels were measured according to the procedures of the CatchPoint Cyclic-AMP Fluorescent Assay Kit (Molecular Devices, Sunnyvale, CA USA). Fluorescence with excitation/emission at 530/590 nm was read with a Flexstation3 plate reader (Molecular Devices).

On day one, SV40-T immortalized neurons were seeded at 70% confluency in a black flat clear bottom 96-well plate (Corning Inc, Kennebunk, ME). On day 3, neurobasal media was removed from the cells and assay buffer, which consists of physiological salt solution (PSS) buffer (126 mM NaCl, 5 mM KCl, 1.2 mM MgCl₂, 10 nM HEPES, 10 nM glucose, 1 nM CaCl₂)+0.2 mM sulfinpyrazone+0.1 mM CaCl₂+12 µl pluronic F-127 (Life Technologies)+60 µg fluo-4 AM (Life Technologies), was added and incubated for 1 h at 37°C and 10% CO₂. Assay buffer was removed and cells were washed once with 1× PBS followed by incubation with PSS buffer+0.2 mM sulfinpyrazone+0.1 mM CaCl₂ for 10 min at 37°C and 10% CO₂. The 96-well plate was inserted into the Flexstation 3 plate reader (Molecular Devices, Sunnyvale, CA) and the cells were treated with 10 µM, 3 µM, 1 µM, 0.3 µM and 0.1 µM of ATP dissolved in PSS buffer. PSS buffer without ATP was included as a negative control. The Flex mode setting was used with λ_ex=494 nm and λ_em=516 nm. ATP-dependent Ca²⁺ response was followed for 120 s over 10 s intervals with six readings per well. The experiment included quadruplicate samples per plate and was repeated two independent times. Dose-dependent curves were fitted and EC50 was calculated.

ATX activity was measured in conditioned media (CM) of WT and ATX-KO B16-F10 cells or BALF from mice injected with WT or ATX-KO B16-F10 cells, as described in [18 (link)]. Briefly, the fluorogenic LPC analog, FS-3 (Echelon Biosciences, Salt Lake City, UT, USA) was used as a substrate for ATX. For measurement of ATX activity in culture medium, CM was collected after 18 h of incubation in serum-free medium, clarified by centrifugation, filtered through a 0.22-μm filter, and concentrated using an Amicon Ultra 30,000 Centrifugal Filter Unit (Millipore; Billerica, MA, USA). For measurement of the activity in biological fluids, BALF was rapidly stored at −80 °C after collection and thawed on ice just before the assay. Five hundred microliters of BALF was then concentrated to 150 μL using an Amicon Ultra 10,000 Centrifugal Filter Unit (Millipore; Billerica, MA, USA). Activity was measured by incubating 30 μL of cell CM or murine BALF with 2 μM FS-3 substrate and 10 μM BSA (Sigma-Aldrich) in assay buffer consisting of 50 mM Tris, (pH = 8.0), 140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, and 1 mM MgCl₂. A 10 nM final concentration of purified recombinant human ATX was used as a positive control. Fluorescence was read every 2 min for 4 h at 37 °C at wavelengths of 485 nm and 538 nm using a FlexStation3 plate reader (Molecular Devices, San Jose, CA, USA).