Poster Presentation 6th Australian Health and Medical Research Congress 2012

Determination of cell proliferation post-irradiation using clonogenic and xCELLigence assays. (#446)

Mohammad Johari Ibahim 1 2 , Premila Paiva 1 , Jeffrey C Crosbie 1 3 , Yuqing Yang 1 , Marina Zaitseva 1 , Peter AW Rogers 1
  1. Department Obstetrics & Gynaecology (Royal Women Hospital), University of Melbourne, Parkville, Victoria, Australia
  2. Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Selangor, Malaysia
  3. Unité INSERM 836 - Medical Beamline, European Synchrotron Radiation Facility, rue Jules Horowitz, Grenoble, France

INTRODUCTION: The Clonogenic survival assay measures radiation effects based on the capability of surviving cells to form colonies. Limitations include variable assay success rates and the long time required to generate a result. Recently a real-time cell-impedance sensing assay was reported showing a good correlation with clonogenic assays when used on irradiated cells (Roa et al, 2011). Synchrotron Microbeam Radiation Therapy (MRT) is an experimental form of radiotherapy which uses synchrotron-generated x-rays collimated to produce intense parallel microbeams, as compared to broad beam (BB) where the radiation is uniform across the targeted area.

OBJECTIVE: To investigate cell response to low, medium or high dose MRT and BB using real time cell impedance measurement compared to clonogenic survival assay.

METHODOLOGY: EMT 6.5 cells (mouse mammary carcinoma) were irradiated with synchrotron MRT (in beam doses: 112, 280 and 560 Gy) and conventional BB (doses: 1.5, 3.8 and 7.5 Gy) (n=3). Clonogenic survival assays were performed and radiation survival fraction curves constructed. For the real-time cell impedance sensing xCELLigence assays, irradiated cells were seeded in 96-well E-plates and impedance measurements recorded every hour.

RESULTS: Clonogenic assays showed that survival fractions of EMT 6.5 cells were decreased with increasing doses of BB and MRT. In contrast, cell indices measured by the impedance assay and taken during the logarithmic growth phase were increased in a dose dependent manner in cells from the same treatment groups

DISCUSSION: The difference between our results and those published by Roa et al, (2011) appear to be due to irradiation stimulating an increase in migratory activity and/or a change in EMT 6.5 cell shape that caused additional attachment to the micro-electrodes resulting in an increased impedance reading and cell index.

CONCLUSION: The cell-impedance assay can be used to demonstrate dose-dependent radiation effects in EMT 6.5 cells, but results contradict those reported previously.