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Article Open Access

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

  • Authors:
    • Júlia Oliveira Dias
    • André Vinícius De Camargo
    • Diego Da Cunha Silveira Alves Da Silva
    • Igor Sampaio Fagundes
    • Laís Bueno Da Silva
    • Mariana De Cássia Bisio
    • Milena Giglioli
    • Rodrigo Gadia
    • Alexandre Arthur Jacinto
    • Wanessa Fernanda Altei
  • View Affiliations / Copyright

    Affiliations: Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo 14784‑400, Brazil, Department of Radiation Oncology, Barretos Cancer Hospital, Barretos, São Paulo 14784‑400, Brazil
    Copyright: © Dias et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY 4.0].
  • Article Number: 73
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    Published online on: June 16, 2025
       https://doi.org/10.3892/wasj.2025.361
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Abstract

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 in vitro, 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 α/β ratio, a key metric for assessing tumor radiosensitivity. However, protocols for fitting curves to the LQ model and calculating α/β 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 ≥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 in vitro.
View Figures

Figure 1

A cell survival curve illustrating a
high α/β ratio compared to another curve with a low
α/β ratio. The blue curve exhibits greater curvature, with a
characteristic shoulder, reflecting a larger contribution from the
β component and a low α/β ratio. By contrast, the red
curve appears more linear, consistent with a high α/β.

Figure 2

Computed tomography images of the
complete setup that were imported to the treatment planning
system.

Figure 3

The setup used for irradiations. (A)
Complete setup from top to bottom, consisting of two 1 cm-thick
solid water plates, a 1 cm-thick gel bolus, a 2 cm-thick custom
thermoplastic support holding a 6-well culture plate, and five
additional 1 cm-thick solid water plates. The total setup height
was 10 cm, with an SSD of 95 cm, so that the cells are at the
isocenter, at a depth of 5 cm in both directions
(anteroposterior/posteroanterior). (B) Open view of the setup,
highlighting the cell culture plate positioned at the isocenter,
and the opening of the radiation field. (C and D) Schematic
cross-sectional illustration of (C) the setup. 1, gel bolus; 2,
custom thermoplastic support; 3, culture plate; 4, solid water
plates. (D) Image alongside the actual setup.

Figure 4

Clonogenic assay using
single-fraction radiation doses. (A) Representative images of the
assay in 6-well plates following a mean incubation time of 6.6
days. (B) Graph depicting the number of colonies counted in each
dose. (C) Survival fractions at different radiation doses. (D)
Survival curve fit to the linear-quadratic model equation (dashed
line), with the Y-axis shown on a logarithmic scale. Plotted values
of colony counts, and survival fractions are displayed above each
bar and error bar. Data represent the mean of sextuplicates (±
standard deviation) from three independent experiments. The
parameters α, β and α/β were calculated using
the linear-quadratic model with a weighted least squares nonlinear
regression. Statistics are relative to the non-irradiated control.
*P≤0.05 and ****P≤0.0001. Ctrl,
non-irradiated control.

Figure 5

Real-time proliferation assay using
single-fraction radiation doses. (A) Optimization of cell seeding
density with an MTS cell viability assay, including four cell
densities across time points indicated by different symbols: 24 h
(circles), 48 h (squares), 72 h (triangles) and 96 h (diamonds)
post-plating. (B) Real-time proliferation curve following
irradiation with 2 Gy (blue) and 8 Gy (red) at 24 h post-plating.
Cell index values were normalized to the highest value observed in
the non-irradiated control, which occurred at 240 h, and expressed
as percentages. (C) Cell index, (D) doubling time, and (E) slope
values for the three conditions. Bars represent the mean values ±
standard error of the mean of quadruplicates from three independent
experiments. Within bars, mean values of cell index, doubling time,
and slope were expressed relative to the non-irradiated control
(1-fold). ****P≤0.0001. Ctrl, non-irradiated
control.

Figure 6

Correlation between surviving
fractions and cell index values in irradiated assays. (A) Cell
index values 6 days after plating and surviving fractions at the
end of the experiment (6 days post-plating). (B) Cell index values
5 days after plating and surviving fractions at the end of the
experiment (6 days post-plating). RTCA, real-time proliferation
assay. CFA, colony formation assay.
View References

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Copy and paste a formatted citation
Spandidos Publications style
Dias JO, De Camargo A, Da Silva DD, Fagundes IS, Da Silva LB, Bisio M, Giglioli M, Gadia R, Jacinto A, Altei W, Altei W, et al: Obtaining cell survival curves in radiobiology: From the linear accelerator to the linear‑quadratic fitting and alternatives. World Acad Sci J 7: 73, 2025.
APA
Dias, J.O., De Camargo, A., Da Silva, D.D., Fagundes, I.S., Da Silva, L.B., Bisio, M. ... Altei, W. (2025). Obtaining cell survival curves in radiobiology: From the linear accelerator to the linear‑quadratic fitting and alternatives. World Academy of Sciences Journal, 7, 73. https://doi.org/10.3892/wasj.2025.361
MLA
Dias, J. O., De Camargo, A., Da Silva, D. D., Fagundes, I. S., Da Silva, L. B., Bisio, M., Giglioli, M., Gadia, R., Jacinto, A., Altei, W."Obtaining cell survival curves in radiobiology: From the linear accelerator to the linear‑quadratic fitting and alternatives". World Academy of Sciences Journal 7.4 (2025): 73.
Chicago
Dias, J. O., De Camargo, A., Da Silva, D. D., Fagundes, I. S., Da Silva, L. B., Bisio, M., Giglioli, M., Gadia, R., Jacinto, A., Altei, W."Obtaining cell survival curves in radiobiology: From the linear accelerator to the linear‑quadratic fitting and alternatives". World Academy of Sciences Journal 7, no. 4 (2025): 73. https://doi.org/10.3892/wasj.2025.361
Copy and paste a formatted citation
x
Spandidos Publications style
Dias JO, De Camargo A, Da Silva DD, Fagundes IS, Da Silva LB, Bisio M, Giglioli M, Gadia R, Jacinto A, Altei W, Altei W, et al: Obtaining cell survival curves in radiobiology: From the linear accelerator to the linear‑quadratic fitting and alternatives. World Acad Sci J 7: 73, 2025.
APA
Dias, J.O., De Camargo, A., Da Silva, D.D., Fagundes, I.S., Da Silva, L.B., Bisio, M. ... Altei, W. (2025). Obtaining cell survival curves in radiobiology: From the linear accelerator to the linear‑quadratic fitting and alternatives. World Academy of Sciences Journal, 7, 73. https://doi.org/10.3892/wasj.2025.361
MLA
Dias, J. O., De Camargo, A., Da Silva, D. D., Fagundes, I. S., Da Silva, L. B., Bisio, M., Giglioli, M., Gadia, R., Jacinto, A., Altei, W."Obtaining cell survival curves in radiobiology: From the linear accelerator to the linear‑quadratic fitting and alternatives". World Academy of Sciences Journal 7.4 (2025): 73.
Chicago
Dias, J. O., De Camargo, A., Da Silva, D. D., Fagundes, I. S., Da Silva, L. B., Bisio, M., Giglioli, M., Gadia, R., Jacinto, A., Altei, W."Obtaining cell survival curves in radiobiology: From the linear accelerator to the linear‑quadratic fitting and alternatives". World Academy of Sciences Journal 7, no. 4 (2025): 73. https://doi.org/10.3892/wasj.2025.361
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