Phosphate buffered saline (pbs)

Laser scanning confocal microscopy was used on a Zeiss LSM700 (Zeiss) confocal microscope equipped with an Axio Observer Z1(Zeiss) inverted microscope using ×100, 1.46 numerical aperture oil immersion lens for imaging. Confocal data were processed using ZEN2010 software (Zeiss). Prior to confocal analysis, 6.2 x 10⁴ MCF-7 cells/petri were seeded in 50 x 7 mm glass petri dishes (WillCo Wells B.V., 1054SE Amsterdam). After 24 h the cells were incubated with green AuNPs (0,46 μg/mL) for 48 h and then exposed to RT at varying doses, with each sample receiving a single dose of either 2.67, 4, 6, 8 and 10 Gy. Then, the cells were washed with PBS (Sigma-Aldrich, Dorset, UK), fixed with 0.25% glutaraldehyde (Sigma-Aldrich, Dorset, UK) for 20 min at room temperature and permeabilized with 0.1% Triton X (Sigma-Aldrich, Dorset, UK) for 5 min. Afterward, the cells were stained first with DAPI (Sigma-Aldrich, Dorset, UK)) and then with Phalloidin in ATTO 488 (Invitrogen; Thermo Fisher Scientific, Inc for the nuclei and actin imaging, respectively).
Morphometric quantification (coherency of F-actin) was performed on 15 cells using the OrientationJ plugin of the ImageJ 1.53a software. This parameter indicates the local orientation of actin filaments. In particular, the value of coherency ranges from 0 (isotropic orientation) to 1 (perfectly oriented structures). Nucleus circularity and nucleus-to-cytoplasm ratios were calculated by ImageJ 1.53a software.

The maturation of cardiomyocytes can be improved
by using substrates with stiffness values similar to those of native
cardiac tissue. In this perspective, different gelatin compositions
(4, 6, 9, and 12% w/v) enzymatically gelled with two different enzyme
concentrations (50 or 100 mg/mL) were prepared and mechanically characterized
by nanoindentation.
Nanoindentation tests were performed at
physiological temperature (37 °C) and in phosphate-buffered saline
(PBS, Sigma-Aldrich, USA) solution in order to mimic the native tissue
conditions, using a Piuma nanoindenter (Optics11, NL) and controlling
the indentation depth (tip radius (r) = 49.5 μm;
cantilever stiffness (K_tip) = 0.48 N/m;
indentation depth (δ) = 5 μm for 4% and 6% w/v gelatin
samples, and δ = 3.5 μm for 9 and 12% w/v gelatin samples).
An area of 3600 μm² was explored for each sample,
setting a scanning step size of 150 μm. To extract the mechanical
properties of the samples, the experimental curves were fitted through
the Hertz model during the first part of the loading phase of the
nanoindentation process. A Poisson’s ratio (υ) of 0.5
was assumed. According to the Hertz contact theory, the applied load
(P) can be expressed as where E* is
the effective modulus, given by where E_sample is
the elastic modulus of the sample and υ’ is the Poisson’s
ratio of the indenter. Assuming that the stiffness of the nanoindenter
tip is infinitely higher than the sample (E_tip ≫ E_sample), the elastic modulus
of the sample can be obtained as
To select a suitable
gelatin composition,
the mechanical characterization
was complemented by a cell culture test for investigating the behavior
of cardiac cells with respect to different compositions. For this
purpose, the different gelatin solutions enzymatically gelled with
two different concentrations of enzymes were poured (192 μL)
in tissue culture plates, and neonatal rat cardiac cells (NRCCs) (∼70%
cardiomyocytes and ∼30% fibroblasts) were seeded on top (6
× 10⁵ cells/cm²) and cultivated for 6 days
in static conditions in a standard incubator (37 °C, 5% CO₂). Functional analysis on the neonatal rat cardiomyocytes
(NRCMs) was performed at the end of the culture by using carbon rod
electrodes placed at a distance of 1.1 cm.

Double emulsion picoreactors were generated from custom fabricated PDMS devices using previously reported designs^{9 (link),18 (link),25 (link),27 (link),30 (link),31 (link)}. All input solutions were loaded into plastic syringes (BD) and connected to the PDMS device via LDPE medical tubing (Scientific Commodities Inc.). Syringe pumps drove the flow of reagents into the device, with standard flow rates of 2×25 μL/hr for two identical inner solutions of PBS (Gibco) with 1% (w/v) Tween-20 (Fisher), 125 μL/hr for the picoreactor fluorocarbon oil with the indicated concentration of dSurf (Fluigent) or PicoSurf (SphereFluidics), and 3500–3700 μl/hr for the outer sheath solution of PBS with 1% (w/v) Tween-20 and 2% (w/v) Pluronic F68 (Millipore Sigma). The PBS inner solution was labeled with 20 μM Fluorescein (Fluka).

Copper sulphate (CuSO₄·5H₂O) and sodium dodecyl sulphate (SDS) were purchased from Himedia Laboratories and Sigma Aldrich, respectively. Europium nitrate (Eu(NO₃)₃·6H₂O), ascorbic acid and sodium hydroxide were obtained from Sisco Research Laboratories Pvt. Ltd. Murine fibroblast cell line (NIH-3T3) and human epithelial cancer cell line (KB cells) were procured from NCCS, Pune. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin and trypsin–EDTA were obtained from Himedia Laboratories. Phosphate buffered saline (PBS) was purchased from Sigma Aldrich.