Section10:Reagents and Buffers for Method Development

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It is critical to ensure the appropriate cell lines expressing the target, control agonist and antagonist standards are available before beginning method development and validation. The minimal requirements are:

1. Transfected cell line with the Gq-coupled hGPCR target. (eg. HEK293, CHO, THP-1 etc.). Receptors coupling through Gi, Go, Gs or Gz can be coupled to Gq via promiscuous G proteins as previously described.
2. Parental cell line control without the target and grown under identical conditions.
3. Agonist, antagonist, and allosteric modulator reference standards (with a wide range of potencies, if available).
4. Poly-D-lysine coated 96- or 384-well plates.
5. Appropriate cell growth media, buffer solutions, trypsinizing reagents.
6. The reagents for ion channels are the same as for GPCRs, with the exception of the FLIPR™ buffer. It is recommended that 5mM calcium be used in the buffer for ion channel experiments. Since HBSS contains 1.3mM calcium, 3.7mM calcium chloride (Sigma) must be added prior to use.
7. Additional reagents needed for a FLIPR assay:
   
   

   Reagent	Manufacturer
   Calcium dyes (Fluo-3, Fluo-4, Calcium 3, Calcium 4, etc.)	Molecular Probes, Molecular Devices
   HBSS	BioWhittaker, Invitrogen
   HEPES	BioWhittaker, Invitrogen
   Probenecid (if needed)	Sigma
   Pluronic Acid	Sigma, Molecular Devices