Clonogenic Assay

Learn that clonogenic assays measure cell colony formation after treatment to assess cancer therapy effects. Carefully label all tubes, wells, and vials with treatment types and controls before beginning.

Treat cells with the test compound (e.g., scorbutic). Remove media, rinse plates, and add 500 µL of 25% trypsin-EDTA solution. Return to incubator for 5-10 minutes to allow cells to detach.

Remove dishes from incubator, add 500 µL fresh media, and pipette to disperse cells evenly. Transfer approximately 50 µL of cells to Eppendorf tubes containing 1.14 mL media, then dilute 1:10 in Isoton solution and count cells using a cell counter.

Draw 900 cells and add to 12 mL media in a conical tube. Mix thoroughly and plate 4 mL per well into six-well culture plates. Incubate for 10-14 days to allow colonies to grow.

Remove plates from incubator and aspirate media. Rinse wells carefully with ethanol sprayed on the side to avoid disrupting colonies. Add fresh ethanol and allow to sit for 5 minutes to fix the cells, then suction off excess ethanol.

Add Coomassie blue to each well, cover to prevent evaporation, and allow to stain for 15 minutes. This dye colors the colonies for easy visualization and counting.

Siphon off excess Coomassie blue and rinse each tray under running water three times until all dye is removed. Allow plates to air dry completely, then count visible colonies to assess treatment effects.