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Criteria
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Biochemical
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Cell-based
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Plate Format*
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96-well or higher density plate
NCGC: 1536-well format
Assay volume 2-6 ul
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96-well or higher density plate
NCGC: 1536-well format
Assay volume 4-6 ul
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Assay Steps
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=10 steps with 96-well plate.
Steps include, reagent additions, timed incubations, plate transfers to incubator, reading, etc.
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=10 steps with 96-well plate.
Steps include, reagent additions, timed incubations, plate transfers to incubator, reading, etc.
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Minimum time increments and maximum assay duration
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Minimum assay window is 5 min. (i.e., earliest time point after last reagent addition)
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<24 hr is ideal; max 48 hrs.
Minimum assay window is 5 min.
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Reagent Addition Steps
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4 maximum (4 unique reagents max; more if pre-mixed)
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4 maximum (4 unique reagents including cells max; more if pre-mixed)
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Reagent removal steps*
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No plate coating steps
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Aspiration steps*
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Temperature
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Between RT and 37°C
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Between RT and 37°C
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Demonstrated DMSO Tolerance*
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0.5 – 1% DMSO
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0.5 – 1% DMSO
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Signal : Background Ratio
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=3-fold
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=3-fold
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Day-to-Day variation of control (e.g., IC50, EC50)
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<3-fold
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<3-fold
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Reagent stability @ final working concentration
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=8 hrs @ RT or on ice bath;
No on-line thawing
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=8 hrs @ RT or on ice bath;
No on-line thawing
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Validation run reagent supply
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10 – 96-well plate equivalents
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10 – 96-well plate equivalents
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Protocol
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Complete detailed protocol. All steps, equipment used, all vendor & catalog # for reagents. Data from 96-well or high density plate tests.
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Complete detailed protocol. All steps, equipment used, all vendor & catalog # for reagents. Detailed cell culture procedure, passage # .Data from 96-well or high density plate tests.
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Detectors
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PE ViewLux (Top reading only: FI, TRF, FP, Abs, Luminescence)
PE Envision (bottom reading FI, ALPHA)
Acumen Explorer (fluorescent laser cytometry)
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PE ViewLux (Top reading only: FI, TRF, FP, Abs, Luminescence)
PE Envision (bottom reading FI, ALPHA)
Acumen Explorer (fluorescent laser cytometry)
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Special
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For unique reagents either investigator prepares sufficient quantity for HTS or identifies a reliable 3rd party vendor.
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Cells must be certified micoplasma-free by direct culture assay and cell-DNA fluorochrome staining.
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Plate Formats: 96-well plates contain 8 rows x 12 columns with volumes ranging between 50-200 ul; 384-well plates contain 16 rows x 24 columns with volumes ranging between 30-50 ul; 1536-well plates contain 32 rows by 48 columns with volumes ranging between 2-8 ul. NCGC will convert assays to 1536-well from 96- or 384-well format.
Reagent removal steps: Are any step that requires the removal of material from the well of a microtiter plate. While such steps may be routine with 96-well plates they are not recommended on robotic systems using 1536-well plates.
Demonstrated DMSO Tolerance: Because all compounds screened are stored in ~100% DMSO and delivered as a 1 to 100 dilution to the assay the assay sensitivity to between 0.5 and 1% DMSO must be determined.
