Spark 20m

NAD⁺ content was measured with an NAD⁺ assay kit following the manufacturer’s instructions (NAD⁺/NADH Quantitation Kit. #MAK037-1KT, Merck, Darmstadt, Germany). The NAD⁺ content of the cells was measured with a plate reader (Tecan Spark 20M, Tecan, Männedorf, Switzerland) at 450 nm.
ATP content was determined using an ATP assay kit following the manufacturer’s instructions (#110M6101, Merck, Darmstadt, Germany). Luminescence was measured with a plate reader (Tecan Spark 20M, Tecan, Männedorf, Switzerland). NAD⁺ content and ATP content were normalized to protein content.

The wound scratch or migration assay was performed with different fibroblast types according to [32 (link)]. Cells were FCS starved for 24 h (as described above). Briefly, the cell monolayer was scraped in a straight line with a 200 µL pipet tip. Along the scratch prefixed points were selected for taking representative images using a phase-contrast microscope (Olympus, Tokyo, Japan) or using a TECAN SPARK 20M (TECAN, Morrisville, NC, USA). Wound closure or cell migration was calculated as the percentage of newly by fibroblasts covered area (3–4images per sample) in relation to the untreated control (taken as 100%). Alternatively, the cell migration was calculated by measuring cell confluence using TECAN SPARK 20M in relation to the untreated control as described above. After scratching, the medium was replaced with fresh media containing different concentrations of FCS or albumin or FCS and AA (100 µM) and cells were incubated for 18–48 h before analysis.

The cell proliferation rate was determined using MTT assay (Promega, Madison, WI, USA). Briefly, cells were plated at 5 × 10³ cells/well in 96-well plates. After adhesion achievement, cells were starved for 24 h without serum and then treated for 24 h with 1% of FBS or 1% of breast cancer sera of MBP-1^-ve and MBP-1^+ve tumors. An amount of 20 μL of the cell titer 96^®AQ_ueous reagent was added to each well after three washes with phosphate buffer saline (PBS) and incubated for 14 h at 37 °C in a CO₂ incubator. The absorbance was recorded at 490 nm using a 96-well plate reader (Spark^® 20M, Tecan Trading AG, Switzerland). The percentage of cell viability was calculated with respect to untreated control cells for each treatment after subtraction of the blank.

A Thermo
Scientific NanoDrop 2000c spectrophotometer equipped with an ultra-micro-cuvette
105.202-QS SD 10 mm from Hellma Analytics was used for UV–vis
spectroscopy measurements. QUANTI-Blue secreted alkaline phosphatase
and MTT assay readouts were performed on a Spark 20M multimode microplate
reader from TecanTrading AG (Mannedorf, Switzerland).

We used P. aeruginosa PAO1 strain or PAO1 (H103) constitutively expressing green fluorescence protein (GFPmut2) [38 (link)] to visualize bacteria in zebrafish embryos. GFP-expressing oprF mutant (H636) was described previously [32 (link)]. Bacteria were grown at 37 °C in Luria broth (LB). Carbenicillin (150 μg/mL) was added for strains expressing GFP. Bacterial growth was monitored in 96-well plates using a Spark 20M (Tecan, Trading AG, Männedorf, Switzerland) microplate reader.

The cytotoxic activity of both NPs-EPS was determined using MTT assay (Promega, Madison, WI, USA), as previously described [69 (link), 72 (link)]. Briefly, cells were plated at 5 × 10³ cells/well in 96-well plates. NPs-EPS, diluted to the desiderated concentrations in culture medium, were added to the wells with respective vehicle control (H₂O). Doxorubicin hydrochloride (Sigma, st. Louis, MO) was used as reference drug. After 24 h of incubation 20 µL of the Cell titer 96^®AQ_ueous reagent was added to each well after three washes with phosphate buffer saline (PBS) and incubated for 1-4 h at 37 °C in a CO₂ incubator. The absorbance was recorded at 490 nm using a 96-well plate reader (Spark^® 20M, Tecan Trading AG, Switzerland). The percentage of cell viability was calculated with respect to untreated control cells for each treatment after subtraction of the blank. The concentration necessary for 50% of growth inhibition (IC₅₀) was calculated using a dose–response model, which was obtained from sigmoidal fitting of response curves of percent inhibition versus logarithmic concentration of DOSs using Graph Pad Prism software. Each result was the mean value of three different experiments performed in triplicate.

Cell viability was assessed using an MTS assay and measured with a Spark 20M multimode microplate reader (Tecan Group Ltd., Mannedorf, Switzerland) at a wavelength of 490 nm. Cell viability = (OD compounds − OD blank)/(OD control − OD blank) × 100% (OD compounds: absorbance value of tested compounds group; OD control: absorbance value of control group; OD blank: absorbance value of culture medium without cells). Cell protection rate = (cell viability of compounds treatment groups—cell viability of model group)/cell viability of model group×100%.