Infinite 200 microplate reader

Mitochondrial membrane potential (MMP) and morphology of the mitochondrial network were determined using rhodamine B hexyl ester and DiOC₆(3). Cells from the early exponential phase of growth were washed twice with sterile water and suspended in a 20 mM HEPES buffer with pH 7.4, containing 5% glucose. Incubation with 100 nM rhodamine B or 175 nM DiOC₆(3) were conducted for 20 min in the dark at 28 °C. After incubation, cells were harvested and resuspended in fresh HEPES buffer. The fluorescence was measured using a TECAN Infinite 200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland) at λ_ex = 555 nm and λ_em = 579 nm for rhodamine B or at λ_ex = 476 nm and λ_em = 501 nm for DiOC₆(3). Mitochondrial network was also visualized using fluorescence microscopy at appropriate wavelengths. The microscopic images, which present typical results from of the duplicate experiment, were captured with the Olympus BX-51 microscope (Olympus, Tokyo, Japan) equipped with the DP-72 digital camera and cellSens Dimension v1.0 software.

The peripheral blood of healthy NSG mice was collected as described above and then diluted with 10 mM PBS (pH 7.4) to obtain a density of 2 × 10⁸ RBC/mL. The diluted RBC (0.5 mL) were mixed with 0.5 mL of Sora-MNC-1 dispersion at various sorafenib doses. RBC dispersed in PBS was used as a negative control, while RBC dispersed in 1% Triton X-100 was used as a positive control. The mixtures were incubated for 1 h at 37 °C and then subjected to centrifugation for 5 min at 1,000×g at 4 °C. After 100 µL of the supernatant was transferred to each well of 96-well plates, absorbance at 576 nm (an indicator of hemoglobin leakage from RBC) was measured using a Tecan Infinite 200 microplate reader (Tecan Group, Switzerland).

Black 384-well
plates were filled with 5 μM protein, and the protein was incubated
with increasing concentrations of adenosine (Sigma-Aldrich), N⁶-methyladenosine (Selleckchem), or two oligoribonucleotides
containing two variants of the m6A consensus sequence, methylated
or unmethylated in A (5′-CCGGm6ACUGUC-3′/5′-CCGGACUGUC-3′;5-′CCGAm6ACUGUC-3′/5′CCGAACUGUC-3,
Dharmacon), in 20 mM Hepes (pH 7.5), 150 mM NaCl, and 10% glycerol.
Fluorescence was measured using a Tecan Infinite 200 Microplate reader
(Tecan Group Ltd.), setting the emission wavelength at 288 nm and
collecting the emission data at 330 nm.

The ferric-reducing ability of plasma (FRAP) assay measures how well a test compound can reduce ferric (i.e., iron-III) to ferrous (i.e., iron-II). Ferric ion (iron-III) is turned into ferrous ion (iron-II) at low pH upon addition of a reducing agent. The formation of ferrous iron can be measured photometrically in a 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) solution since iron (II) forms a coloured complex with TPTZ [31 (link)].
Following addition of the to-be-tested compound (or the vehicle control) to an iron (III) chloride solution (1.7 mM) with TPTZ (1.67 mM) in acetate-buffered solution (228 mM) at pH 3.6 and 15 minutes of incubation, absorbance at 620 nm was measured. The absorbances resulting from ferrous ion/TPTZ complex formation were plotted against the concentrations of the tested compound.
FRAP and ABTS measurements were carried out in a Tecan Infinite 200 microplate reader (Tecan Group Ltd., Crailsheim, Germany). The final concentrations of the flavonoids and vitamins measured were 645, 323, 161, 65, 32, and 0 (solvent control) nM. In order to calculate the gradient relative to trolox, linear regressions were carried out and the gradient from the plotted flavonoid/vitamin curve was divided by the trolox gradient.
All experiments were carried out a minimum of three times (different days).

Total phenolic content was determined as gallic acid equivalents (GAE) using the Folin–Ciocalteu reagent [27 ]. The macroalgae phenolic extract (50 µL) was diluted with deionized water (750 µL), Folin-Ciocalteu phenol reagent (50 µL) was added, and contents were mixed thoroughly. After 1 min, 150 µL of 20% sodium carbonate solution was added, followed by thorough homogenization. After incubation for 1 h at 37 °C, absorbance was measured at 760 nm using a Tecan infinite 200 Microplate Reader (Tecan Trading AG, Switzerland). The measured value was compared to a standard curve prepared with a gallic acid solution. Total phenolic content was expressed as GAE per gram of dried extract.

The cell viability was evaluated by MTT assay with slight modifications [41 (link)]. Caco-2 cells were cultured in 96-well plates at 1.2 × 10⁴ cells per well and allowed to adhere for 24 h. Then, the cells were washed with fresh medium and treated. After 24 h of incubation, 10 μL of MTT (5 mg/mL) was added for 4 h at 37 °C. The medium were removed carefully, followed by the addition of 150 μL of DMSO to each well to dissolve the precipitate. The absorbance was then measured at 570 nm using a Tecan infinite 200 microplate reader (Tecan Trading AG, Switzerland). Cell viability was calculated by Equation (4) where A_t is the absorbance of treated cells and A₀ is the absorbance of control.