Pharmacology JoVE (Open Access) Citable · DOI

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Name: Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms
Uploaded: 2026-06-17
Duration: 8 min 46 s
Description: Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

DOI: 10.3791/52879-v

What you'll learn

✓ Establish GI50 values and initiate drug-resistant cell line derivation
✓ Isolate single-cell clones from drug-resistant populations
✓ Assess and characterize acquired drug resistance mechanisms

Protocol

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

Difficulty

advanced

Total time

~4–8 weeks (includes iterative drug exposure and clonal selection)

Model organism

Cell culture (cancer cell lines)

Biosafety

BSL-1

Steps

Assess GI50 and initiate drug resistance assays

Determine GI50 (growth inhibition at 50%) for compounds of interest using dose-response curves, then establish drug-resistant cell lines by chronic or escalating drug exposure in vitro.

▶ 01:01

Isolate single-cell clones from resistant populations

Derive monoclonal cell lines from drug-resistant populations using single-cell cloning techniques to ensure genetic homogeneity and eliminate residual sensitive cells.

▶ 04:27

Assess and validate acquired drug resistance

Confirm resistance phenotype through re-testing GI50 values in isolated clones and prepare samples for downstream sequencing to identify molecular resistance mechanisms.

▶ 06:37

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