Clonogenic assay

Learn that the clonogenic assay measures the reproductive potential and proliferation ability of cells after treatment with drugs or ionizing radiation. Understand that a single clonogenic cell divides to form a colony, and colonies are measured using colony forming units (CFU).

Assemble sterile materials including growth medium with serum (fetal bovine serum or BSA), cell culture flasks, petri dishes, 0.25-0.5% trypsin, and non-sterile materials including PBS, formaldehyde, crystal violet, and a hemocytometer or electronic cell counter.

Expose monolayer cell cultures to the experimental agent (ionizing radiation or chemical drug) for 24-48 hours, with exposure time depending on the toxicity effects of the specific compound being tested.

Detach treated cells using 0.25% trypsin, then perform serial dilutions and plate cells according to the plating efficiency to establish appropriate seeding densities.

Place plated cells in a CO₂ incubator for 9-14 days and observe cell growth daily under a microscope to monitor colony formation and development.

Fix cells with 4% formaldehyde followed by PBS washes, then stain colonies with 1% crystal violet solution to visualize and count the surviving colonies.

Measure the number of colonies formed and calculate plating efficiency by dividing colonies by seeded cells. Then determine survival fraction by dividing experimental colonies by seeded cells and correcting for plating efficiency to assess the effect of treatment.

Create a semi-log plot showing survival fraction versus experimental agent concentration. Identify IC₅₀ (concentration inhibiting 50% colony survival) and IC₉₀ (concentration inhibiting 90% colony survival) to quantify treatment effects and discuss factors influencing the assay.