Spectramax paradigm multi mode

FITC-MSN and FITC-HA-MSN were administrated through tail vein injection (200 μL, 5 mg/kg) separately, and then cheek blood was taken quickly at 1 min, 30 min, 1 h, 6 h, 12 h, 24 h, and 48 h. Afterward, 50 μL of blood was mixed with an equal volume of PBS each time, and then the blood was quantitatively transferred to a 96-well plate. The fluorescence intensity was measured by a microplate reader (SpectraMax Paradigm Multi-mode, Molecular Devices, Sunnyvale, CA, USA) at an excitation wavelength of 490 nm and an emission wavelength of 530 nm.

Antibiofilm activity was determined by microtiter-plate technique [21 (link)] with crystal violet (CV) assay, counting viable cells in Colony Forming Units (CFU) [22 (link)]. In order to determine the PAEO and carvacrol action in the biofilm, plates were subjected to optical density microplate reading (Molecular Devices -SpectraMaxParadigmMulti-Mode) at a wavelength of 570 nm. Activity was found in concentrations that failed to display the adhesion of crystal violet in any of the wells.

The viability of cells was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. 10,000 HUVECs or 6000 A549 cells were seeded into each well on a 96-well plate. After incubation for 24 h, the medium was replaced with the fresh medium containing the NBP@TiO₂ nanostructures at different concentrations. After 48-h treatment, the medium was discarded, and a fresh medium (100 μL) containing MTT (0.5 mg mL⁻¹) was added into each well. After incubation for 3 h, the medium was discarded and the resultant formazan crystal was dissolved with dimethyl sulfoxide (150 μL). The absorbance of each well was measured using a SpectraMax Paradigm multimode microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 540 nm. The cell viability for each sample relative to control was calculated.
Calcein acetoxymethyl ester (calcein AM) staining was also used to determine the cell viability. After the treatment, the medium was replaced with a fresh medium containing calcein AM (2 μM, Thermo Fisher Scientific). After incubation for 30 min at 37 °C, the cells were washed with a fresh medium. The green fluorescence of the cells was observed under an Olympus IX71 fluorescence microscope.

The levels of formic acid in collected urine samples were determined by the Formate Assay Kit (ab111748, Abcam, Cambridge, UK), following the protocol from the manufacturer. Ten microliter of urine was used per well, and the absorbance at 450 nm was measured on a 96-well microplate reader (SpectraMax Paradigm Multi-Mode, Molecular Devices, San Jose, CA, USA). The concentration of formic acid was calculated according to the standard curve.

Approximately 2.5×10⁵ amebic transformants were incubated in BIS medium containing 2 mg/ml fluorescent fluid-phase marker RITC dextran at 35°C for indicated time points. The cells were collected, washed three times with PBS, and resuspended in 250 μl of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Triton-X 100). Fluorescence intensity was measured using a plate reader (SpectraMax Paradigm Multi-Mode, Molecular Devices, California, USA) at an excitation wavelength of 570 nm and an emission wavelength of 610 nm.
Approximately 2×10⁴ amebic transformants were incubated in BIS containing 20 μM CellTracker Blue (Thermo Fisher) at 35.5°C for 1 hr. After staining, approximately 10⁴ amebic transformants resuspended in 100 μl of BIS medium were transferred to a well on a 96-well glass-bottom plate. After incubation at 35.5°C in an anaerobic chamber for 40 min, 0.5 mg/ml of transferrin conjugate with Alexa Fluor 568 was added to the well and images were acquired by CQ1 and analyzed as above.

Microtiter-plate technique [11 (link)] with the crystal violet (CV) assay was used [10 (link)]. For determination of EOPH action in the biofilm, the plates were subjected to reading the optical density using a microplate reader (Molecular Devices-Spectra Max Paradigm Multi-Mode) at a wavelength of 595 nm. The activity was found in concentrations that did not observe the adhesion of crystal violet in any of the wells.