title: Optimized digital counting colonies of clonogenic assays using ImageJ software and customized macros: comparison with manual counting.
abstract: OBJECTIVE
  To develop a digital method for counting colonies that highly replicates manual counting.
  
  
  METHODS
  Breast cancer cells were treated with trastuzumab-conjugated gold nanoparticles in combination with X-ray irradiation, (111)In labeled trastuzumab, or γ-radiation, followed by clonogenic assays. Colonies were counted manually or digitally using ImageJ software with customized macros. Key parameters, intensity threshold and minimum colony size, were optimized based on three preliminary manual counts or blindly chosen. The correlation of digital and manual counting and inter- and intra-experimenter variability were examined by linear regression. Survival curves derived from digital and manual counts were compared by F-test (P < 0.05).
  
  
  RESULTS
  Using optimized parameters, digital counts corresponded linearly to manual counts with slope (S) and R(2) value close to 1 and a small y-intercept (y(0)): SK-BR-3 (S = 0.96 ± 0.02, R(2) = 0.969, y(0) = 5.9 ± 2.2), MCF-7/HER2-18 (S = 0.98 ± 0.03, R(2) = 0.952, y(0) = 0.74 ± 0.47), and MDA-MB-231 cells (S = 1.00 ± 0.02, R(2) = 0.995, y(0) = 3.3 ± 4.5). Both reproducibility and repeatability of digital counts were better than the manual method. Survival curves generated from digital and manual counts were not significantly different; P-values were 0.3646 for SK-BR-3 cells and 0.1818 for MCF-7/HER2-18 cells. Using blind parameters, survival curves generated by both methods showed some differences: P-values were 0.0897 for SK-BR-3 cells and 0.0024 for MCF-7/HER2-18 cells.
  
  
  CONCLUSIONS
  The colony counting using ImageJ and customized macros with optimized parameters was a reliable method for quantifying the number of colonies.
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cai
authors.forename: Niladri
authors.initials: N
authors.lastname: Chattopadhyay
authors.forename: Wenchao Jessica
authors.initials: WJ
authors.lastname: Liu
authors.forename: Conrad
authors.initials: C
authors.lastname: Chan
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Pignol
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Reilly
mesh_terms: D001943:Breast Neoplasms / Q000473:pathology / Q000532:radiotherapy; D002138:Calibration; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005260:Female; D006801:Humans; D016014:Linear Models; D012984:Software; D014410:Tumor Stem Cell Assay / Q000379:methods*