title: Cell density dependent plating efficiency affects outcome and interpretation of colony forming assays.
abstract: OBJECTIVE
  The usefulness of colony forming assays (CFA) has been established for almost 40 years (Puck and Marcus, J.Exp.Med. 103: 653-666, 1956). Although time-consuming and not successful for all cell lines, it is generally considered to be the gold standard of assays for testing the sensitivity of cell lines to ionizing radiation or other cytotoxic agents in vitro. We recently found for several cell lines that the plating efficiencies of both control and irradiated cells is dependent upon the density of cells seeded for colony formation; that is, increasing cell inoculum levels resulted in a non-linear relationship with colony formation, even at relatively low colony numbers.
  
  
  METHODS
  All data from a human melanoma cell line, transfected with c-myc or N-ras, as well as from normal human diploid fibroblasts, were taken to see how this phenomenon influenced outcome and interpretation of clonogenic assays. Survival was recalculated using all data, or only data with a linear relationship between inoculum level and colony formation.
  
  
  RESULTS
  It is found that when data with a non-linear relationship between inoculum level and colony formation are included, survival can be underestimated due to inhibition of colony formation in treated cultures.
  
  
  CONCLUSIONS
  For validity, colony forming assays must be standardized to assure a constant relationship between the cell density and colony forming efficiency. This usually requires a much lower density of colonies than has been typically published for many cell survival studies.
authors.affiliation: Department of Clinical Oncology, University Hospital, Leiden, The Netherlands. POMP@rullf2.LeidenUniv.nl
authors.forename: J
authors.initials: J
authors.lastname: Pomp
authors.forename: J L
authors.initials: JL
authors.lastname: Wike
authors.forename: I J
authors.initials: IJ
authors.lastname: Ouwerkerk
authors.forename: C
authors.initials: C
authors.lastname: Hoogstraten
authors.forename: J
authors.initials: J
authors.lastname: Davelaar
authors.forename: P I
authors.initials: PI
authors.lastname: Schrier
authors.forename: J W
authors.initials: JW
authors.lastname: Leer
authors.forename: H D
authors.initials: HD
authors.lastname: Thames
authors.forename: W A
authors.initials: WA
authors.lastname: Brock
mesh_terms: D000818:Animals; D016466:CHO Cells; D002470:Cell Survival / Q000528:radiation effects; D003114:Colony-Forming Units Assay / Q000379:methods*; D006224:Cricetinae; D005347:Fibroblasts / Q000528:radiation effects; D006801:Humans; D008545:Melanoma / Q000473:pathology; D014410:Tumor Stem Cell Assay / Q000379:methods