a density of ~30,000 cells per well in Hams-F12 medium, supplemented as above and cultured overnight. The cells were then incubated at 37C for 60 minutes with the appropriate calcium indicator; Fluo-3, Fura-2 or FlexStation Calcium Assay Kit; and 2.5 mM probenecid (see appendix). The cells loaded with Fluo-3 or Fura2 were washed manually (BioHit 8-channel multipipette) three times with Hanks BSS medium containing 2.5 mM probenecid. Cells loaded with the FlexStation Calcium Assay Kit did not
require this wash step. The final volume in each well was 200 mL.
The cell plates were then placed into the FlexStation system to monitor fluorescence before and after the addition of ATP (50 ml addition, ATP made at 5X final concentration, final concentrations 30 nm-30 mM). Experimental parameters are tabulated below.
Responses were measured as peak fluorescence intensity minus basal fluorescence intensity (Fluo-3 or FlexStation calcium reagent) or the 340/380 nm excitation ratios, calculated within SoftMax Pro, for Fura-2. Data are expressed as mean s.e. mean and n > 12.
Individual sets of E/[A] curve data were fitted to a four-parameter logistic of the form:
where α, β, EC50 and m are the asymptote, bottom of curve, location (expressed as -log10 EC50 ) and slope parameters respectively. [A] is the concentration of the agonist where, in this case, its ATP. E is the relative fluorescence units or ratio of fluorescence units for ratiometric dyes. All curve-fitting routines were carried out using GraphPad Prism 3.0 (GraphPad Software, Inc.).
Z-factors were calculated using the formula:
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where αc+ denotes standard deviation of the positive control and αc- represents standard deviation of the ne
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