title: Agarose overlay selectively improves macrocolony formation and radiosensitivity assessment in primary fibroblasts.
abstract: OBJECTIVE
  Primary fibroblasts are not suitable for in vitro macrocolony assay due to their inability to form distinct colonies. Here we present a modification of agarose overlay that yielded extensive improvement in their colony formation and assessment of radiosensitivity.
  
  
  METHODS
  Macrocolony formation was assessed in primary human fibroblasts VH10 and HDFn with or without overlay using 0.5% agarose in growth medium at 24 h post-seeding. Malignant human cell lines (A549, U87) and transformed non-malignant fibroblasts (AA8 hamster, MRC5 human) were used for comparison.
  
  
  RESULTS
  Agarose overlay caused significant improvement marked by early appearance (one week) of distinct colonies with high cell density and multifold higher plating efficiency than conventional macrocolony assay in VH10 and HDFn human fibroblasts. Compared to conventional assay or feeder cell supplementation, agarose overlay resulted in broader cell morphology due to improved adherence, and yielded more compact colonies. Gamma-radiation dose-response survival curves could be successfully generated for both fibroblast cell lines using this method, which yielded no such effects in the transformed/malignant cell lines tested.
  
  
  CONCLUSIONS
  This easy and inexpensive 'agarose overlay technique' significantly and selectively improves the fibroblast plating efficiency, thus considerably reducing time and effort to greatly benefit the survival studies on primary fibroblasts.
authors: Natural Radiation Response Mechanisms Group, Division of Radiation Biosciences, Institute of Nuclear Medicine & Allied Sciences (INMAS) , Delhi , India.
authors: Sudhir
authors: S
authors: Chandna
authors: Raghubendra Singh
authors: RS
authors: Dagur
authors: Ankit
authors: A
authors: Mathur
authors: Adayapalam Tyagarajan
authors: AT
authors: Natarajan
authors: Mats
authors: M
authors: Harms-Ringdahl
authors: Siamak
authors: S
authors: Haghdoost
mesh_terms: D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D005347:Fibroblasts / Q000166:cytology* / Q000528:radiation effects*; D006801:Humans; D011836:Radiation Tolerance; D012685:Sepharose
chemical_list: D012685:Sepharose