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Colony Formation Assay severe

Plated too dense — colonies merge into one another

Symptom
Many colonies fuse together so individual colonies cannot be counted reliably; counts will be over- or under-estimated.
Common Causes
  1. 1 Seeding density was too high for the well/dish size
  2. 2 Cells were not dispersed into single-cell suspension before plating
Solutions
  1. 1 Lower the seeding density and run a pilot to find the right cell number per well
  2. 2 Trypsinize thoroughly and pipette to ensure single-cell suspension before plating
  3. 3 Plate so each potential colony is clearly separated
Related Video (3)
Bilibili (China-Accessible Mirrors) ★ 82
Colony Formation Assay — Step-by-Step Protocol (Bio-Techne)
"Explicitly covers seeding density optimization and colony counting, matching the failure's root cause and counting problem."
YouTube (Curated Tutorials) ★ 78
A Guide to the Colony Forming Cell Assay: Methods and Tips
"Step-by-step CFC protocol with tips that directly address consistent performance and seeding-related issues."
YouTube (Curated Tutorials) ★ 72
Clonogenic Cell Survival Assay (Colony Formation Assay)
"Detailed colony formation assay walkthrough useful for correctly performing the technique and understanding the context."
Source: xiaohongshu.com ↗
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