4.6. Clonogenic Assay

Clonogenic assay was performed to compare the two groups: GBM cells treated with GNPs (100 μg/mL) versus GBM cells alone. Experiments were carried on a 6-well plate. At 80% confluence, 3 × 10⁴ cells were seeded per well for 24 h. Next, they were incubated with the GNPs (100 μg/mL) overnight. Subsequently, they were irradiated the following day. Radiation was delivered using single doses: 2, 4, 6, and 8 Gy using a LINAC. Immediately after irradiation, 2000 cells were seeded in each well plate and incubated at 37 °C with 5% CO₂ for 10 days or until colonies of greater than 50 cells had been formed. After sufficient colonies had formed, DMEM was removed, washing performed three times with 1 mL PBS, and 500 µL of 10% (v/v) neutral-buffered formalin with 50 µL of crystal violet added to each well for 60 min at room temperature. Next, the formalin–crystal violet mixture was removed with repeated washing using deionized water. Colonies of 50 cells or more were counted manually and surviving fractions were calculated, dividing the plating efficiency of GNP (100 μg/mL) treated cells by plating efficiency of GBM alone (No GNP). The experiment was repeated for an additional two times to have a total of three independent experiments (n = 3). Survival fraction (SF) was calculated based on the following equations [32]:

where PE is the plate efficiency. All the PEs of the treated samples were normalized to that of the control non-irradiated plates.

Survival fraction (SF) results were fit with a linear-quadratic (LQ) model, represented by Equation (2). Data was generated using GraphPad Prism 8.0 and plotted on a log (% survival) vs. dose plot.

where S is the survival fraction, α and β are the model constants, and D is the absorbed dose in Gy.