Obtaining cell survival curves in radiobiology: From the linear accelerator to the linear‑quadratic fitting and alternatives

Radiobiology is the study of the biological effects of ionizing radiation on living organisms. A fundamental aspect is the use of cell survival curves (CSCs) to evaluate radiosensitivity <em>in vitro</em>, with the clonogenic assay being the gold standard method for this purpose. Despite its widespread use, this assay has inherent limitations, such as variability in cell plating efficiency, and the need to adjust cell seeding density based on the expected cell kill at higher radiation doses. This issue is often overlooked in published protocols, potentially compromising reproducibility and robustness. Survival data from clonogenic assays can be fitted to the linear‑quadratic (LQ) model to determine the <em>&alpha;/&beta;</em> ratio, a key metric for assessing tumor radiosensitivity. However, protocols for fitting curves to the LQ model and calculating <em>&alpha;/&beta;</em> values are poorly explained, lacking standardized protocols in the literature. Modern approaches, such as impedance‑based real‑time cell analyses (RTCAs) to monitor cell proliferation provide an alternative for obtaining CSCs, providing dynamic monitoring of cell proliferation and survival after irradiation. The present study demonstrates a fully described and reproducible protocol with step‑by‑step instructions for obtaining survival curves from clonogenic assays and fitting them to the LQ model. The present study also demonstrates a strong correlation (R² &ge;0.9222) between RTCA results and clonogenic assay data, emphasizing potential of RTCA as a high‑throughput, sensitive and more rapid alternative for assessing radiosensitivity <em>in vitro</em>.