sarcoma ATCC HTB-96; ATCC, USA) cells were
grown in Dulbecco’s modified Eagle’s medium
(DMEM)/F-12 supplemented with 10% fetal
improvements in physiochemical properties for this
series will need to be realized in order to access tool
compounds suitable for in vivo experimentation.
bovine
serum
and
2mM
L-glutamine.
Recombinant FFA4 (NM_001195755) expressing
cells were generated by transducing U2OS cells
with BacMam viruses encoding the respective
receptor and the chimeric G-protein Gα16
according to established protocols. In brief, cells
were plate to a density of 2x105 cells/mL in cell
culture medium containing human FFA4 (0.25%,
v/v), mouse FFA4 (0.5%, v/v) or rat FFA4 (0.8%,
v/v) BacMam virus. Gα16 BacMam virus
(0.12%, v/v) was also transduced in preparation of
recombinant human FFA4 cells to allow efficient
coupling of human FFA4 receptor to
phospholipase C pathway. This solution of
cells/virus mixture was then plated at a density of
104 cells/well and cultured at 37°C, 5% CO2, 95%
humidity for 24h. Functional EC50 Studies cells
were performed in cells incubated with Hank’s
buffered salt solution (HBSS) containing the
cytoplasmic calcium indicator, Fluo-4 dye in the
acetylmethyl form (4mM), 2.5mM probenecid
and 250µM Brilliant Black at 37 °C for 60 min.
Compound plates were generated containing 3%
dimethyl sulfoxide in dye loading buffer.
Compounds (i.e. FFA4 agonist GSK137647A or
Histamine for host untransduced U2OS cells)
were added to the cells at a 1:3 dilution and
calcium mobilization was measured using a
Fluorescence image plate reader (FLIPR,
Molecular Devices). Data were converted into
normalized responses with respect to assay
standards GSK137647A (for FFA4) or Histamine
(for Host U2OS cells). Data were further analyzed
using a 4-parameter fit to calculate EC50 values.
(b) Condreay, J. P.; Witherspoon, S. M.; Clay, W.
C.; Kost. T. A. 1999. Proc. Natl. Acad. Sci. 1999,
96, 127.
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17. Verified via quantitative RT-PCR from RNA
isolated from MIN6 and NCI-H716 cell lines
analyzed using FFA4-specific primer probe sets.
11. (a) Assay Conditions: U2OS (human osteo-