Parafilm m

For clonogenic X‐ray irradiations, five dose points were investigated in triplicate, 0, 1.5, 3, 4.5, and 6 Gy, chosen to decrease the survival fraction by approximately two orders of magnitude to give a sufficient cell survival curve fitting for each cell line and nanoparticle combination, whereas the DNA damage samples were irradiated with 1 Gy. Before irradiating with X‐rays, the dishes were filled with medium, providing adequate scattering conditions. The dishes were then sealed using Parafilm M (Sigma‐Aldrich, UK) immediately before irradiation. Irradiations were carried out at the National Physical Laboratory, Teddington, UK, with a 6 MV linac (Elekta Versa HDTM), with a dose rate of 6.5 Gy min⁻¹. Dishes were placed in the center of a 10 × 10 cm² field (Figure9), where dose calculations were based on reference conditions using depth dose data from ionization chamber measurements for this field size. The dose output was also confirmed with ionization chamber measurements traceable to the UK primary standard. The beam uniformity was assessed using Gafchromic EBT3 film (Ashland ISP Advanced Materials, NJ, USA), where the dose variation across the sample was less than 2%.

MS grade water (H₂O), acetonitrile (ACN), methanol (MeOH), ethanol (EtOH) and chloroform were purchased from Biosolve (Dieuze, France). The cleavable detergent ProteaseMAX was purchased from Promega (Charbonnieres, France). Parafilm M, 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (HCCA), aniline, sodium dodecyl sulfate (SDS), dl-dithiothreitol (DTT), trifluoroacetic acid (TFA) and formic acid (FA) were purchased from Sigma (Saint-Quentin Fallavier, France).

To assess the volume and area of the repaired tissue and calculate the tissue and bone mineral density, formalin-fixed samples were visualised by means of microcomputed tomography (SkyScan 1172, Bruker, Billerica, MA, USA) at 8 µm voxel size after the 24-h rehydration in 0.9% NaCl solution. To maintain normal hydration during the scanning, bones were wrapped in the Parafilm M (P7543, Sigma-Aldrich, St. Louis, MO, USA). Calibration of bone mineral density was performed using Hounsfield units. For the calibration, we used HA samples with 8 mm diameter and 0.25 or 0.75 g/cm³ mineral density. In contrast to bone mineral density, tissue mineral density was defined as a density specific to the mineralised tissue and therefore excluding any surrounding non-bone tissue. Scanning was carried out with 0.75 mm aluminium filter to reduce beam hardening. Semiquantitative image analysis was performed utilising the CTAn software (Bruker, Billerica, MA, USA).

DISH was performed using a DNA-specific dual-color probes ZytoDot2C SPEC HER2/CEN 17 Probe Kit (ZytoVision, Bremerhaven, Germany). Sections were deparaffinized and incubated first for 5 min in 3% H₂O₂ to block endogenous peroxidase, and then for 15 min in a prewarmed pretreatment EDTA solution at 95–100 °C in a water bath. After washing the sections in distilled water, 100 μl of pepsin solution was applied to the slides, which were then incubated for 5 min at room temperature in a humidity chamber. Thereafter, the sections were washed in distilled water and dehydrated through an ethanol series. Ten microliters of the ZytoDot 2C SPEC HER2/CEN 17 Probe (P) was applied to each slide, after which the sections were covered with a coverslip and sealed with Parafilm^®M (SIGMA, USA). The samples were then denatured at 80 °C for 5 min, transferred to a humidity chamber and hybridized overnight (16–18 h) in a hybridization oven at 37 °C. After hybridization, immunodetection was performed according to the manufacturer’s instructions, and the sections were counterstained with hematoxylin and mounted. HER2 expression was assessed by examination under a microscope.

The CyQuant GR assay (Sigma-Aldrich, UK) was used to determine the number of attached MSCs at 1, 3, and 24 h post seeding on TCP, P, SLA, and modSLA discs. The kit consists of a fluorescent dye that exhibits enhanced fluorescence on intercalating with double-stranded nucleic acids of ruptured cells and was used according to the manufacturer's instructions. For this experiment, human MSCs (N = 2) were expanded as described above. Cells were detached from culture flasks and then seeded at a density of 1 × 10⁴ cells per surface in 1 ml of serum-free basal medium (alpha-MEM, Gibco, UK). Adherence of MSCs to each surface was evaluated with three replicates (n = 3) per cell line. At each time point, samples were gently washed twice with PBS and lysed by ×3 freeze-thaw cycle that alternated between -80°C and room temperature, during which the samples in 24-well plates (Nunc) were firmly enclosed using sealing film (Parafilm M, Sigma-Aldrich, UK). A working solution of dye was then added at 150 μl per well for 10 min at 4°C in the dark. A 100 μl volume of homogenate per sample was transferred to an opaque 96-well plate (Nunc) for fluorescence intensity measurement at excitation 530 nm and emission 590 nm (Fluoroscan, Tecan, Switzerland). Total cell numbers were determined by interpolating fluorescent intensities of samples from a standard curve.

For the analysis of stability, NO-heme was stored in 15 ml centrifuge tubes (Greiner Bio-One, Frickenhausen, Germany) under different light and atmospheric conditions. Briefly, 80 ml of the NO-heme solution were first synthesized, purified and concentrated as described above. The final solution was further diluted in 80% acetone/H₂O to a final volume of 12.5 ml and allocated to four centrifuge tubes containing 3 ml each. Two of the NO-heme samples were stored in the dark at room temperature, flushed with nitrogen and additionally sealed with thermoplastic film (Parafilm^® M, Sigma-Aldrich). The other samples were incubated at room temperature and exposed to sunlight and air. The NO-heme content of each tube was analyzed by UV–Vis and FTIR spectroscopy after 0, 2, 4 and 24 h of incubation.

Mosquitoes were artificially fed with different diets: (1) 10% sucrose (ad libitum), (2) rabbit blood, (3) bicarbonate-buffered saline-agarose (BBSA) supplemented with Serratia marcescens (Sm). The BBSA solution was composed of glucose (10 mg), 500 mM freshly made bicarbonate buffer pH 7.4 (10 μL), 0.5 mg low melting-point agarose and 100 mM ATP, pH 7.4 (5 μL). The final volume was set to 500 μL with 150 mM NaCl. Feeding was performed using water-jacketed artificial feeders maintained at 37°C and sealed with Parafilm “M” (Sigma-Aldrich, St. Louis, MO) membrane. For depletion of mosquito’s microflora, females were fed with sucrose 10% supplemented with antibiotics, penicillin (100 u/mL) and streptomycin (100 μg/mL) (LGC biotecnologia, Cotia, SP) for 4 days, as previously described [23 (link)].