Spark microplate reader

For measuring proteasome peptidases activity, dissected heart tissue or isolated PBMCs were lysed on ice in a 26S proteasomes isolation buffer as previously described [6 (link)]. Protein content was adjusted with Bradford, and supernatants were immediately used (after the addition of the fluorogenic substrate) to determine the CT-L proteasomal peptidase activity; all measurements were performed in duplicates. Emitted fluorescence was recorded at Spark^® Tecan microplate reader (Tecan Group Ltd., Maennedorf, Switzerland) a VersaFluor Fluorometer System (Bio-Rad Laboratories, Hercules, CA, USA) at excitation and emission wavelengths of 380 nm and 460, 350, and 440 nm, respectively. Results are expressed as % ± SEM fold change of 13–14-week-old C57Bl/6J mice used as control samples, referred to as young adult mice in the manuscript.

MDA-MB-231 were plated in 96-well plates at a density of 15 × 10³ cells/well. After 24 h, cells were treated with unloaded or Dox-loaded nanocages for different times at 37 °C. Time and dose-dependent toxicity of free Dox were assessed on MDA-MB-231 and HeLa cells (Figure S5). The MTS assay was performed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Spark microplate reader (Tecan Trading AG, Männedorf, Switzerland) was used for measuring the absorbance at 492 nm. Cell viability of treated cells was normalized to the control condition (untreated cells).

CFS-treated pathogen viability was assessed with BacTiter-Glo^TM Microbial Cell Viability Assay (Promega Italia S.r.l., Milan, Italy). Pathogens were seeded at OD₆₀₀ = 0.01 (approximately 5 × 10⁶ CFU/mL) into a 96-well-plate, immediately treated with probiotic CFSs (50% v/v) and then incubated at 37 °C in static conditions. A plate for each pathogen and each time point of 24, 48, and 72 h was used. The viability assay was then performed following the manufacturer’s instructions and the luminescence was detected with a Spark microplate reader (Tecan Trading AG, Switzerland). A complex viability assay with pathogen co-culture was also optimized. Pathogens were plated altogether at the same OD₆₀₀ = 0.01 and allowed to adapt for 1 h at 37 °C before CFS treatment. Then, the assay was executed as described above. In all the experiments, TSB, iMRS, and iCysMRS were used as controls. Each experiment was done with five replicates and repeated three times independently.

A curcumin stock solution was prepared by dissolving curcumin powder in DMSO at a 10 mg/mL concentration. Curcumin stock solutions in the hydrogels were fixed to 0.05 wt % for all hydrogels. Curcumin stock solution was added to the double-distilled water before the addition of PS and/or FmocFF. The release of curcumin from the hydrogels was determined in simulated body fluid (SBF). The hydrogels were immersed in 5 mL SBF at 37 °C on an orbital shaker at 100 rpm. Aliquots of SBF were taken at predetermined time points, 100 µL of the SBF supernatants were placed in a black opaque 96-well microplate. The curcumin released from the hydrogels was measured using a Spark microplate reader (Tecan Trading AG, Switzerland) [21 (link)]. Emission at 520 nm was recorded with an excitation wavelength of 420 nm. Released curcumin concentrations were determined using a standard curve constructed by recording the absorbance of serial dilutions of curcumin solution of known concentrations. Spectra were corrected by subtraction of the corresponding buffer signal. The cumulative release of curcumin was expressed and plotted over time.

Cell lysis and ELISA procedures were performed following the manufacturer’s instructions (PathScan® Phospho-p44/42MAPK (Thr202/Tyr204), Cell signaling Technology, USA). The cells were lysed with lysis buffer (Cell Signaling Technology, USA), and the supernatants of the lysed cells were used for ELISA. The cell lysates of the samples were incubated in the phospho-p44/42MAPK (ERK1/2)-coated 96-well plate overnight at 4°C. Afterward, the sequential incubation with the detection antibody, the HRP-linked secondary antibody, and TMB substrate was for 1h, 30 min, and 10 min at 37°C, respectively. Generous washing (4 times) with wash buffer was performed after each step using an automated plate washer (Bio-Tek Instruments, USA). Finally, the assay reaction was terminated by adding stop solution and reading the absorbance at 450 nm using Spark microplate reader (Tecan Trading AG, Switzerland).

Dissolution studies were performed with a Copley’s Dissolution Test Dis8000 (Nottingham, UK). Samples were sunk in 500 mL of 0.1 M phosphate with 0.05 M sodium lauryl sulphate buffer (pH 7.40 ± 0.05) kept at a constant temperature of 37.0 ± 0.1 °C and constantly stirred at 50 rpm. Then, 5 mL of the sample solution was withdrawn at regular intervals and filtered via 0.45 µm MF-millipore membrane filter (MILLEX HA). An equivalent amount of fresh buffer kept at the same temperature was reintroduced. All dissolution tests were done in triplicate. Pulsatile release tablets represent the only exception to this method; in this case, the samples were collected every 15 min for the entire duration of the experiment and the buffer solution (125 mL) was completely removed and substituted with a fresh one after every sampling session. This again was performed in triplicate.
The amount of drug released into the medium as a function of time was obtained by analysing the collected samples using UV-visible spectrophotometry, a Spark Microplate Reader (Tecan Trading AG Männedorf, Switzerland) UV/Vis Spectrophotometer equipped with a deuterium—tungsten light source. The λ_max = 290 nm peak of maximum absorbance for Fenofibrate was thus used as a reference in the analysis of all samples. All results were analysed using OriginPro.