( buffer was prepared as described on th...)Gq-coupled Receptor Assays Using FLEXstation

Reagent Preparation
1. Prepare a fresh stock of 250 mM probenecid. Dissolve 710 mg of probenecid in 5 mL of 1M NaOH and then add 5 mL of 1X Reagent Buffer.

2. Add 1:50 dilution of 250 mM probenecid to the Loading Buffer (final concentration, 5 mM) and 1:100 dilution of 250 mM probenecid to 1X Reagent Buffer (final concentration, 2.5 mM). Probenecid inhibits endogenous efflux pumps that would prevent the dye from accumulating inside the cell.

3. Prepare a fresh stock of 5 mM ADP in 1X Reagent Buffer containing 2.5 mM probenecid. Prepare dilutions of ADP with 1X Reagent Buffer containing 2.5 mM probenecid (see Table 1).

Dye Loading of Cells
1. Add 100 L of Loading Buffer containing 5 mM probenecid to 100 L of cells that were plated the night before (final volume, 200 L/well).

2. Incubate cells for 0.5 h at 37 C.

3. In the meantime, prepare compound plates.

Compound Plate Preparation
Fifty microliters of ADP was added by FLEXstation to the 200 L already in the assay plate well. Since this was a 1/5 dilution, 5X stocks of ADP were made, starting with a 5 mM stock, which was subsequently diluted in series at 1:3 in 1X Reagent Buffer (see Table 1). Dead volume for the V-bottomed compound plates is less than 10 L, so keep this is mind when deciding the volume of compound to add. Each row of the compound plate contained 12 replicates of one concentration of ADP. Row A contained 1X Reagent Buffer alone (negative control), and rows B through H contained threefold dilutions of 1 mM ADP beginning with 1 mM ADP in row B as shown in Table 1.

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