High-Throughput Screening Assay Guidance Criteria
| Criteria | Biochemical | Cell Based |
|---|---|---|
| Plate Format* | 96-well or higher density plate 1536-well format Assay volume 2-6 μl | 96-well or higher density plate 1536-well format Assay volume 4-6 μl |
| Assay Steps | = 10 steps with 96-well plate. Steps include, reagent additions, timed incubations, plate transfers to incubator, reading, etc. | = 10 steps with 96-well plate. Steps include, reagent additions, timed incubations, plate transfers to incubator, reading, etc. |
| Minimum time increments and maximum assay duration | Minimum assay window is 5 min. (i.e., earliest time point after last reagent addition) | < 24 hr is ideal; max 48 hrs. Minimum assay window is 5 min. |
| Reagent Addition Steps | 4 maximum (4 unique reagents max; more if pre-mixed) | 4 maximum (4 unique reagents including cells max; more if pre-mixed) |
| Reagent removal steps* | No plate coating steps | Aspiration steps* |
| Temperature | Between RT and 37°C | Between RT and 37°C |
| Demonstrated DMSO Tolerance* | 0.5 - 1% DMSO | 0.5 - 1% DMSO |
| Signal: Background Ratio | = 3-fold | = 3-fold |
| Day-to-Day variation of control (e.g., IC50, EC50) | < 3-fold | < 3-fold |
| Reagent stability @ final working concentration | = 8 hrs @ RT or on ice bath; No on-line thawing | = 8 hrs @ RT or on ice bath; No on-line thawing |
| Validation run reagent supply | 10 - 96-well plate equivalents | 10 - 96-well plate equivalents |
| Protocol | Complete detailed protocol. All steps, equipment used, all vendor and catalog # for reagents. Data from 96-well or high density plate tests. | Complete detailed protocol. All steps, equipment used, all vendor and catalog # for reagents. Detailed cell culture procedure, passage # .Data from 96-well or high density plate tests. |
| Detectors | PE ViewLux (Top reading only: FI, TRF, FP, Abs, Luminescence) PE Envision (bottom reading FI, ALPHA) Acumen Explorer (fluorescent laser cytometry) | PE ViewLux (Top reading only: FI, TRF, FP, Abs, Luminescence) PE Envision (bottom reading FI, ALPHA) Acumen Explorer (fluorescent laser cytometry) |
| Special | For unique reagents either investigator prepares sufficient quantity for HTS or identifies a reliable 3rd party vendor. | Cells must be certified micoplasma-free by direct culture assay and cell-DNA fluorochrome staining. |
Table Notes:
Plate Formats: 96-well plates contain 8 rows x 12 columns with volumes ranging between 50-200 μl; 384-well plates contain 16 rows x 24 columns with volumes ranging between 30-50 μl; 1536-well plates contain 32 rows by 48 columns with volumes ranging between 2-8 μl. 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.
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