Counting clonogenic assays from normoxic and anoxic irradiation experiments manually or by using densitometric software.
The clonogenic assay is the method of choice to determine cell reproductive death after in vitro irradiation treatment. Traditionally, colony quantification has been performed by manual counting, a very laborious, time-consuming and rather subjective task. In this study, we compared manual counting by two skilled investigators with automated counting using the freely available ClonoCounter program. Five human tumour cell lines were irradiated under normoxia (21% O(2)) or anoxia (<0.1% O(2)), after 24 h or 6 h anoxic preincubation. Colonies were quantified manually or using the ClonoCounter software. A positive correlation between the absolute number of colonies counted manually and automatically was shown. Though there was a general trend of underpredicting the absolute number of cell colonies when counting automatically, survival curves were very similar, and in none of the cell lines were significant differences in radiobiological parameters such as mean inactivation dose, surviving fraction at 2 Gy and oxygen enhancement ratio detected. Our results suggest that the ClonoCounter provides sufficient reliability to be implemented for counting human tumour colonies in in vitro irradiation experiments. In contrast to several previously reported computer-aided colony-counting methods, it is a freely available program, requiring only minimal instrument costs.
Wouters
An
A
Laboratory of Cancer Research and Clinical Oncology, Department of Medical Oncology, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. An.Wouters@ua.ac.be
Pauwels
Bea
B
Lambrechts
Hilde A J
HA
Pattyn
Greet G O
GG
Ides
Johan
J
Baay
Marc
M
Meijnders
Paul
P
Lardon
Filip
F
Vermorken
Jan B
JB
D000818:Animals; D002452:Cell Count / Q000379:methods*; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D018384:Oxidative Stress / Q000528:radiation effects*; D011836:Radiation Tolerance; D012984:Software; D012986:Software Validation; D014410:Tumor Stem Cell Assay / Q000379:methods*