The cytotoxicity of Pt NFs was evaluated performing colony formation assays (CFA), which determine cells survival by measuring the ability of a single cell to proliferate and form a colony. Briefly, HeLa cells were seeded in T-25 ventilated culture flasks (BioLite 25 cm2 cell culture treated flask, Thermo Scientific) at a density of 2 × 105 cells per flask. They were incubated 6 or 12h with enriched medium containing Pt NFs at three different Pt molar concentrations: 2.5 × 10−4, 5 × 10−4 and 10−3 mol L−1. HeLa cells incubated with an equal volume of ultrapure water were use as control. Following incubation, cells were harvested and re-seeded into petri dishes at different densities to yield approximately 100 surviving colonies per dish. After 2 weeks, the obtained colonies were fixed, stained and manually scored. The analysis of variance was performed using the ANOVAOneWay method provided by OriginPro8.5.
Biolite 25 cm2 cell culture treated flask
Manufactured by Thermo Fisher Scientific
The BioLite 25 cm2 cell culture treated flask is a laboratory equipment product designed for cell culture applications. It provides a surface area of 25 cm2 for cell growth and proliferation. The flask is treated to enhance cell attachment and adhesion.
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2 protocols using biolite 25 cm2 cell culture treated flask
1
Cytotoxicity Evaluation of Platinum Nanoflakes
2
Clonogenic Assay for Cell Survival
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Cell survival was assessed by a standard 14 days colony formation assay (CFA). Exponentially grown HeLa cells (2×105) were plated in T25 culture flasks (BioLite 25 cm2 cell culture treated flask, Thermo Scientific) and maintained overnight in a 5% CO2 incubator at 37°C. Cells were then incubated for 6 h with enriched culture medium containing PtPEG NPs at a final Pt concentration of 5×10−4 mol.L−1. An equivalent water volume was added into control samples. Afterwards, the cells were irradiated in fresh medium under atmospheric conditions at room temperature, independently by γ-rays (137Cs) and C6+ ions. Immediately after exposure, the cells were trypsinized and re-seeded into 10 cm Petri dishes (Falcon 3002) at different densities in order to achieve ca. 100 colonies per dish. Formed colonies were fixed and stained with a 0.5% methylene blue 50% (v/v) methanol solution. The colonies containing more than 50 cells were scored and the calculated survival fractions were normalized to the survival fraction of the corresponding control. The HeLa plating efficiency was found close to 65%.
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