An aequorin protocol provides an alternative for cell-based screening using calcium mobilization (Figure 1). Aequorin assays are easier to automate as they do not require cell wash steps, and optimal assay performance can be achieved at room temperature. Using cells in suspension renders aequorin assays more amenable to automated operation in 1,536-well format with increased throughput and productivity. The use of cell suspensions provides reduction in cell plating, and maintenance costs and sensitivity of the assay afforded by high signal-to-background means fewer cells per well can be used than in fluorescent-based assays.
Assay costs are further reduced using the low-cost reagent coelenterazine (<$.02/well) which has little cellular toxicity and is not subject to efflux. Reduced compound interference arising from auto-fluorescence and toxicity should produce fewer false positives and a cleaner data set.
Chinese Hamster Ovary cells expressing recombinant histamine H1 receptors and mitochondrial-targeted aequorin (CHO-Aeq-H1: EuroScreen) were grown in the following media: DMEM:F12 (1:1), 1% Penicillin-Streptomycin, 2 mM L-glutamine, 10% fetal calf serum (FCS), and 500 g/mL geneticin. Cells were routinely cultured at 37C in an atmosphere of 95% air/5% CO2.
On the day of assay the cells were harvested using versene and resuspended at a density of 5x106 cells/mL in DMEM (without phenol red) supplemented with 0.1% BSA and 15mM HEPES medium. Active aequorin was reconstituted by the addition of 5M coelenterazine h (Invitrogen C 6780) and the cell suspension stirred at 200 rpm on a magnetic stirrer for four hours in the dark at room temperature.
Prior to measurement (384-well format) on LumiLux, the cell suspension was diluted 10-fold and transferred to the integrated cell stirrer assembly. For agonist assays, cell suspension (25 l) was dispensed onto compound (25 l) in a 384-well black, clear-bottom plate (Greiner) with simultaneous imaging of the entire plate for 40 seconds with 10-second baseline prior to cell addition. For antagonist assays, compound and cells were pre-mixed (25 l of each), incubated for 10 minutes, with a final addition (25 l) of agonist (EC80).
The 1,536-well format agonist assays were conducted with 3 l of the cell suspension dispensed onto compound (3 l) in a 1,536-well black, clear bottom LoBase plate (Greiner). For the antagonist assay compound and cells were pre-mixed (3 l of each), incubated for 10 minutes, with a final addition (3 l) of agonist (EC80).