A Guide to the Colony Forming Cell Assay: Methods and Tips

Thoroughly shake the methylcellulose-based media bottle to mix, allow air bubbles to escape, then use a sterile 14-gauge needle and 5 ml syringe to aliquot the appropriate volume into sterile tubes. Store aliquots at -20°C in a manual defrost freezer until use.

Thaw methylcellulose aliquots at room temperature for 30-60 minutes. Simultaneously resuspend cells in pre-warmed media and centrifuge at 400×g for 5 minutes. Discard supernatant and resuspend the cell pellet in a small volume of fresh media.

Count viable cells using a hemocytometer with 0.4% trypan blue staining. Calculate the total cell number needed based on recommended plating densities for human or mouse assays, adjusting the cell suspension to the desired concentration.

Add the appropriate volume of cell suspension to the methylcellulose aliquot and vortex vigorously. Allow air bubbles to escape at room temperature for 15-30 minutes, then distribute 1.1 ml of the cell-media mixture to each 35 mm petri dish or 6-well plate using a sterile 16-gauge needle.

Gently rotate and tap each plate to spread the media evenly, then remove remaining air bubbles with a pipette tip. Place petri dishes in larger plates containing sterile water, or fill void spaces in 6-well plates with sterile water to prevent drying during culture.

Place plated cells in a 37°C incubator with 5% CO₂ and maintain culture for 13-16 days for human cells or 6-9 days for mouse cells. Avoid disturbing plates during incubation to prevent colony shifting.

After incubation, count resulting colonies using a printed grid or manual markings to stay oriented and avoid recounting. Identify colony types (BFU-E, CFU-E, CFU-M, CFU-G, CFU-GM, CFU-GEMM) based on characteristic morphology, color, and cell size differences observed under the microscope.