Microreader

Cell viability was evaluated by ATP assay with 4T1 cells. Briefly, 4T1 cells with an initial density of 5000 cells/well were seeded in a 96-well plate. After 24 h’s inoculation, the incubation medium was aspirated and replaced with RPMI solutions containing different concentrations of GNWs or GNSs. After another 24 h’s incubation, incubation medium was aspirated. 50 µL ATPlite 1 step substrate solution was mixed with 50 µL RPMI medium and the mixture was added into each well. The plate was sealed and mixed for 10 min at room temperature before test. Luminescence signals were measured using a microreader (Biotek). Average luminescence intensity was computed and compared.
For treatment with nanoparticles + X-ray, 4T1 cells with an initial density of 5000 cells/well were incubated in 96-well plates. 24 h after the inoculation, cell medium was replaced with 100 µL RPMI medium solutions containing GNWs or GNSs. 5 Gy X-ray radiation was applied 24 h after the incubation. 24 h after the irradiation, cell medium was replaced with a mixture of 50 µL of ATPlite 1 step substrate solution and 50 µL RPMI. The plate was sealed and mixed for 10 min at room temperature before test. Luminescent signals were measured on a microreader (Biotek). Average luminescence intensity was computed and compared.

SOSG assay was performed by following vendor’s protocol (ThermoFisher). Typically, 100 µg SOSG was dissolved in 33 µL methanol to make a 5 mM stock solution. The solution was diluted with Milli-Q water to 5 µM test solution before use. 4T1 cells were incubated in 96-well plates with an initial density of 5000 cells/well. 50 µg/mL in 100 µL incubation medium was added after 24 h’s inoculation. After another 24 h’s of incubation, 5 Gy X-ray radiation was applied. Immediately after irradiation, cell medium was aspirated and replaced with 100 µL RPMI containing 5 µM SOSG. The plate was kept in the dark at room temperature. Fluorescence signals were measured on a microreader (Biotek). Excitation/emission wavelength were set at 504/525 nm. Similar protocol was used for SOSG studies with nanoparticle solutions.

Total RNA was isolated from cells by using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), following the manufacturer’s instructions. The total RNA concentration was measured with a microreader (BioTek Instruments Inc., Winooski, VT, USA) at 260/280 nm. Afterwards, 1 μg of total RNA was dissolved in 80 μL of 0.1% diethyl pyrocarbonate treated-deionized water for cDNA synthesis. The total RNA had a final volume of 20 μL. The SYBR Green PCR Master Mix kit (Life Technologies, Carlsbad, CA, USA) and Mx3000P qPCR System (Agilent Technologies, Santa Clara, CA, USA) were used to perform quantitative real time polymerase chain reaction (qRT-PCR). The reaction mixture comprised 1 μL of cDNA, 10 μL of 2× SYBR Green Master Mix, and the appropriate forward and reverse primers in a final volume of 20 μL. The PCR conditions were as follows: 95°C for 30 min, followed by 40 cycles of 95°C for 5 s, 60°C for 20 s, and 72°C for 20 s. The primers are listed in Table 1. Fluorescent signals were measured at the annealing/extension step, and each sample from each group was analyzed in duplicate for each tested gene. The qRT-PCR reaction was performed on a thermal cycler (C1000; BIO RAD, Hercules, CA, USA). The results of qRT-PCR were analyzed by 2^−ΔΔCT method.