Enspire multimode

NK cell cytotoxicity was evaluated by lanthanide (Europium, Eu) fluorescence assay. Briefly, target cells were labeled with BATDA (PerkinElmer) for 30 minutes at 37°C in the complete medium. Labeled cells were transferred to 96-well polystyrene plates (U-bottom, Nunc), mixed with NK cells at different E/T ratios, and incubated for 2 hours at 37°C. For ADCC, NK cells were incubated with SKOV-3 target cells in the presence of 50 ng/mL of either anti-HER2 antibody or control IgG. After cytolysis, the cell supernatant, containing the released TDA ligand, was added to the Eu solution to generate the fluorescent Eu-TDA chelate. The fluorescence of Eu-TDA was measured using EnSpire multimode plate reader (PerkinElmer). The amount of released TDA ligand in cell supernatants was regarded as the experimental TDA release. Total TDA release was measured after complete lysis of target cells by 1% NP-40. Lysis percentage was calculated using the following equation: (experimental TDA release – spontaneously released TDA) / (total TDA release – spontaneously released TDA).

To measure the fluorescence for plasmid assay, the cells were grown in LB with shaking, and culture time-point samples were collected in 20–30 min intervals during the exponential phase. Cells were gently pelleted, washed once with PBS buffer and resuspended again in 600 μl of PBS buffer. One third of each sample was used to monitor the optical density (600 nm) of bacteria, and other two thirds to read the green fluorescence (msfGFP) intensity (emission at 515 nm with an excitation at 485 nm) and red fluorescence (mKate2) intensity (emission at 633 nm with an excitation at 588 nm) in a 96-well plate reader (EnSpire Multimode; Perkin Elmer). Relative fluorescence was corrected by subtracting the level of fluorescence of non-fluorescent bacteria cells and dividing by the optical density. Arbitrary units were obtained by determining the slope of a plot of fluorescence versus culture density via linear regression. This approach both ensures that cells are in pseudo-steady-state, and provides greater precision than single-time-point assays.

Twelve selected cytokines and growth factors (VEGF, PDGF-AA, PDGF-BB, HGF, FGF-basic, EGF, TGF-β1, IL-1α, IL-1β, IL-7, IL-10, and TNF-α) were measured in skin extracts and culture medium using a LEGENDPlex custom made kit (Biolegend, San Diego, CA, USA). In this bead-based multiplex immunoassay, each bead is marked by a defined amount of allophycocyanin (APC) and conjugated with a capture antibody specific for a target protein. After incubation with skin extracts or medium, the addition of detection antibodies and streptavidin-phycoerythrin (SA-PE) generates a unique fluorescence signal for each target protein, which is in proportion to the antigen concentration. Each sample and standard were run in duplicate. The fluorescence intensity was detected by flow cytometry (FACS Calibur, BD, Germany). At least 400 events in the gated region were measured. The concentration of each analyte was calculated using LEGENDplex software (https://www.biolegend.com/legendplex/software, last access on 27 December 2021). The analyte concentration in skin extracts was normalized to the total protein content, which was assessed by the Pierce 660 nm Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA). Absorbance values were determined using a plate reader (EnSpire Multimode Perkin Elmer, Akron, OH, USA). The data are shown as increments of each analyte over time.

The ORAC-FL analyses were carried out on a plate reader (EnSpire multimode Perkin-Elmer, Waltham, MA, USA) of 96 wells of white polystyrene. The fluorescence was read from the top, with an excitation wavelength of 485 nm and an emission of 528 nm. The reaction was carried out in 75 mM (pH 7.4) phosphate buffer. 150 µL of fluorescein solution (FL) (40 nM, final concentration) and 25 µL of the extract and nanoparticles were added to each well of the plate. The sample was incubated for 7 min at 37 °C and then 25 µL of a 2,2′-azo-bis(2-methylpropionamidine) dihydrochloride (APPH) solution (Sigma-Aldrich 97%, St. Louis, MO, USA) 18 mM final concentration, were added. Fluorescence was recorded every minute for 120 min and it was used as FL and AAPH control, using solvent instead of the solution of the extracts [48 ,49 (link),50 (link)]. A Trolox (3 μM to 20 μM) calibration curve was prepared as a reference antioxidant. The inhibition capacity was expressed as μmol Trolox equivalent per gram of extract. All reaction mixtures were prepared in triplicate and at least three independent assays were performed for each sample. The area under the fluorescence decay curve (ABC) was calculated by integrating the fluorescence decrease.

Cells were seeded at 7.5 × 10⁵ cells per well in a 6-well plate and grown overnight (12 h). After removing the media and washing cells, PBS (250 µL) was added, and cells were incubated for 15 min. PBS was then collected in ice-cold microcentrifuge tubes and spun at 300× g for 2 min at 4 °C. Supernatants were transferred to a black 96-well plate. ADP was included to ensure the selectivity of the assay. ADP was measured using an ADP assay kit (Abcam) according to the manufacturer’s instructions. Fluorescence was measured at Ex/Em 535/587 nm using a plate reader (EnSpire Multimode, PerkinElmer^®, Waltham, MA, USA).