96 well black polystyrene plate

The ORAC values were determined by M5 according to the method [2 (link)]. Fluorescence (225 μL per well, 8.163 ∗ 10⁻⁸ mol/L) was added into 96-well black polystyrene plate (Costar), followed by adding sample, blank, or trolox (30 μL per well) and incubated at 37°C for 20 min. AAPH (25 μL, 0.36 M) was added to the mixture and measured every minute for 2 h immediately (excitation wavelength 485 nm and emission wavelength 535 nm). Different concentrations of trolox were used as a standard (12.5 μg/mL, 25 μg/mL, 35 μg/mL, 50 μg/mL, and 75 μg/mL) to measure ORAC value of EECP and EEPG. The results were expressed as the trolox equivalent (mmol) per gram of propolis.

GO powder (1–10 mg/mL) was dissolved in DI water and sonicated
for at least 30 min until a homogeneous solution was obtained. DNA-decorated
GO sensor was prepared by adding 5 μL of 2 μM fluorescence-labeled
probe to 5 μL of GO solution (1–10 mg/mL). Mixing was
performed by repetitive pipetting several times. GOs and fluorescence-labeled
probes were incubated in the dark for 10 min. Subsequently, the sample
was adjusted to a final volume of 100 μL with 1× phosphate-buffered
saline solution and transferred to a black 96-well plate (Costar;
96-well black polystyrene plate). The final concentrations of GOs
and fluorescence-labeled probes were 50–500 μg/mL and
100 nM, respectively. For sensitivity testing, 5 μL of the RCA
product from various concentrations of target miRNA was introduced
to the optimal concentration of the DNA-decorated GO sensor (10 mg/mL)
and was further incubated in the dark for 30 min before being transferred
to a black 96-well plate. The fluorescence response of the sample
was measured using a fluorometer (SpectraMax, Molecular Devices, USA)
at 640/670 nm for the F-21 complex (Cy5) and 492/520 nm for the F-16
complex (FAM).

To evaluate the metabolic activity of cells exposed to NP or NP@Dox, the Deep Blue Cell Viability kit was used according to the manufacturer’s instructions. Briefly, cells were incubated for 24, 48, and 72 h in the presence of the NP, NP@Dox, or the free drug. Afterward, the culture medium was removed, cells were washed with PBS, and a solution of 10% (v/v) of reagent in the culture medium was added and incubated for 4 h at 37 °C in a humidified atmosphere with 5% CO₂; 100 μL of each sample was loaded into a Costar^® 96-Well Black Polystyrene Plate and the fluorescence values were read at excitation and emission wavelengths of 530 and 590 nm, respectively, using a microplate reader (Synergie HT, Bio-Tek). Data are expressed as a percentage related to the CTR condition.

Cells (2.0 × 10⁴ cells/well) were plated into 96-well black polystyrene plates (Corning®, NY, USA) maintained in a humidified atmosphere at 37°C and 5% CO₂. Cells were stimulated for 4 h and 24h with SF from 16-h and 36-h of single and dual-species biofilms. Following the stimulation, AlamarBlue^TM reagent (Invitrogen^TM, Thermo Fischer Scientific Inc., MA, USA) was added (20 μl/well) and incubated for 4 h at 37°C. For the control of living cells, the cells were grown under standard conditions in RPMI-1640 medium (processed in the same way as the analyzed samples). For the cell death control, the cells were treated with Triton^TM X-100 (Sigma-Aldrich®, St Louis, MO, USA). Fluorescence measurements were performed on Fluoroskan Ascent® FL (Thermo Fisher Scientific Inc., MA, USA) equipment with 544-nm excitation filters and 590-nm emission filters. The assay was performed in triplicate on at least three separate occasions. The fluorescence measurements were converted to percentages based on the control without a stimulus. Non-normal values were analyzed using the Shapiro-Wilks test. In addition, the Kruskal-Wallis test was applied along with the Dunn's Multiple Comparison Test using GraphPad PRISM vs 8.0 software.

Saturation binding curves for protein-peptide interactions were determined by examining the change in fluorescence polarization (FP). The GluK1 signal peptide was synthesized with a fluorescein isothiocyanate (FITC)-labeled at N-terminal. Peptide probes (100 nM) were used in the titration with increasing amount of GST-fusion proteins in 100 μl FP buffer (100 mM KCl, 20 mM HEPES (pH 7.4)) in 96-well black polystyrene plates (Corning). The plate was read on a Synergy H1 microplate reader (BioTek) in the fluorescence polarimeter mode to acquire polarization values (P). The resulting P values were plotted against the concentration of proteins after subtraction of baseline. To determine the K_d value, a curve was fitted by the equation Y = B * X/(K_d + X), with B being the maximum P value that would be reached at saturation as indicated by the extrapolation of the fitted curve.

HPAEC grown to confluence in a 96 well black polystyrene plates (Corning), were subjected to adhesion assay as described^{35 (link)}. HL-60 cells were labelled with 2.5 µM Calcein-AM (Invitrogen) for 30 minutes. 200 µl of Calcein-AM loaded HL-60 cells were added to each well and incubated for 1 hour at 37 °C. After 1 hour the non-adherent cells were removed by gentle wash with Phenol Red free DMEM media. Fluorescence was measured using a fluorescent plate reader at an excitation of 495 nm as described^{35 (link)}.