COMPOUND HANDLING
Dissolve compounds in appropriate vehicle (DMSO for most small molecules, PBS for biologics). Final DMSO concentration should not exceed 0.1-0.5% to avoid solvent toxicity. Prepare fresh dilutions for each experiment, include vehicle controls, and verify compound stability in culture media over treatment duration.
DOSE-RESPONSE DESIGN
- Concentration Range: 8-10 point half-log dilution series spanning 4-5 log units
- Starting Point: Base on plasma Cmax from clinical data or prior screens
- Replicates: Minimum n=3 biological replicates for IC50/EC50 determination
- Controls: Vehicle (negative), known toxic/active compound (positive)
- Exposure Time: Acute (24h), subchronic (7 days), chronic (14-28 days)
TOXICITY ENDPOINTS
- Viability: ATP content (CellTiter-Glo), MTT/MTS, live/dead imaging
- Apoptosis: Caspase 3/7 activation, Annexin V staining, TUNEL
- Functional: Albumin/urea (liver), contractility (heart), barrier (gut)
- Morphology: Organoid size, structure integrity, differentiation markers
- Biomarkers: LDH release, ALT/AST, troponin, KIM-1
DATA ANALYSIS
Fit dose-response curves using 4-parameter logistic regression. Report IC50/EC50 with 95% confidence intervals. Compare to clinical exposure data using therapeutic index (TI = TC50/Cmax). Include historical data for reference compounds to validate assay performance across experiments.