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CCK-8 Cell Viability Assay moderate

High well-to-well variability — poor reproducibility

Symptom
Parallel wells in the same group give widely different OD values; SD bars are very large.
Common Causes
  1. 1 Pipetting was uneven
  2. 2 Cells settled unevenly across the plate
  3. 3 Edge effect from evaporation
  4. 4 Incubation times differed across wells
  5. 5 Insufficient mixing or air bubbles in wells
Solutions
  1. 1 Pipette gently and accurately, avoiding bubbles
  2. 2 After seeding, gently rock the plate to distribute cells evenly
  3. 3 Fill outer rows with sterile PBS to reduce edge evaporation
  4. 4 Add CCK-8 reagent at the exact same time across all wells
  5. 5 Mix gently after adding reagent
Related Video (2)
YouTube (Curated Tutorials) ★ 92
CCK-8 Operation Guide & Data Analysis
"Directly demonstrates CCK-8 operation protocol and highlights critical procedural details, including pipetting technique which is the root cause of the well-to-well variability failure."
YouTube (Curated Tutorials) ★ 78
Cell Counting Kit-8 (CCK-8) Assay Principle
"Explains CCK-8 assay principle and how to use the kit correctly, providing foundational knowledge on proper technique to minimize variability."
Source: xiaohongshu.com ↗
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