Biotek synergy ht microplate reader

Cells from the previous step were lysed in a high-pressure homogenizer (Stansted Fluid Power Ltd., London, England) (one pass, 20 bar) under sterile conditions. Cell debris was removed from the lysate by centrifugation (10,000× g, 4 °C, 15 min). Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instructions. The collected RNA was purified using RNA purification columns (Thermo Fisher Scientific Inc., Waltham, MA, USA) following the manufacturer’s instructions.
Sample degradation and integrity were monitored by agarose gel electrophoresis (1% w/v, 100 mV, 30 min). The purity and recovery yield of RNA were calculated using the absorbance values measured at 260 and 280 nm using a UV spectrophotometer (BioTek Synergy HT Microplate Reader; BioTek Instruments, Inc., Winooski, VT, USA).

To assess cellular GSH and GSSG levels and their ratio fluorometric GSH/GSSG Ratio Detection Assay Kit (Abcam, Cambridge, UK) was used according to the manufacturer’s protocol with lysate obtained as described above. Briefly, the sample cell lysate or GSH, or GSSG standards were added to the thiol green indicator reaction mixture and were incubated for 60 min in room temperature and protected from light. Then an increase in fluorescence was monitored at Ex/Em = 490/520 nm with Bio-Tek Synergy HT Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA). GSH and GSSG standards were used to create a graph of the standard curve. Fluorescence in blank wells (with the assay buffer only) was used as control and was subtracted from the values for those wells with the GSH reactions. Reduced GSH and total GSH were measured through calculation form standard curves and the GSSG amount was calculated as a difference between total GSH and reduced GSH.

The encapsulation efficiency (EE) and the loading capacity (LC) of the three tested natural compounds in the SLNs were determined indirectly. To separate the free compounds from the loaded SLNs, the samples were diluted in ultrapure water (1:200) and subsequently filtered (Amicon Ultra Centrifugal Filters Ultracell-30 kD, Merck Millipore Ltd., Burlington, MA, USA Tullagreen, Carrigtwohill; from Sigma-Aldrich) at 14,000× g for 3 min. After the centrifugation, the filtrate containing the free compounds was collected and mixed with ethanol 100% (v/v) at a volume ratio of 3:7. The amount of free natural compound was individually quantified by UV-Vis absorbance at the characteristic wavelength of each compound (274 nm for EGCG, 305 nm for resveratrol and 377 nm for myricetin) (BioTek Synergy HT Microplate Reader, BioTek, UK). Previously, the maximum wavelength of each compound was determined, and a calibration curve was drawn for each compound. The concentration of free compounds was determined from a calibration curve in ethanol 100% (v/v) at a volume ratio of 3:7 (water). This experiment was run in triplicate for each type of loaded-SLNs and the EE was determined as described in the following equation (Equation (1)).

The LC was determined following the equation below (Equation (2)).

HT-29, SW620, MDA MB 231, MCF7, A549, and H1299 were seeded at a density of 3 × 10⁴ cells/well in 96-well plates. The cells were grown overnight and then followed by transfections. After 24 h, fresh media were applied to replace the transfection reagent-containing media with 11 mM of docetaxel or vehicle, then the cells were incubated for an additional 24 h. The Apo-ONE Caspase-3/7 Assay Kit (Promega Corporation, Madison, WI, USA) was used to measure apoptosis according to the protocol described by the manufacturer. Then, 100 µL of homogeneous Caspase-3/7 reagent was added to each well and the plate incubated at room temperature for 30min to 2h. Fluorescence was detected for measuring (at wavelengths of 499/521 nm excitation/emission) the apoptosis state (Caspase 3/7 activity) using Biotek Synergy HT Microplate Reader (BioTek, Winooski, VT, USA).

U937 cells were purchased from the American Type Cell Collection. The cells at 2–3×10⁶ cells/ml were grown in RPMI 1640 medium (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO). Fresh U937 cells were then incubated with 10 µM Calcein AM (BD Biosciences) at 37°C for 1 h with 5% humidified CO₂. Subsequently, an aliquot of U937 cells (∼1×10⁶ cells/ml) suspended in serum-free RPMI 1640 medium was added to the upper compartment of the 24-well BD Falcon HTS FluoroBlok Inserts (BD Biosciences) [25] (link). This apparatus has a polyethylene terephthalate (PET) membrane (8 µm pore size) that blocks the transmission of light from 490 to 700 nm. This allows detection of cells present in the lower compartment only. The cells were allowed to migrate into the lower compartment at 37°C for 2 h in the presence of _pE-MCP1-6His, with the recombinant _Q-MCP1 (PeproTech) as a negative control. Once cells migrate through the pores of the PET membrane, they are no longer shielded from the light and can be detected by a fluorescence plate reader (Bio-Tek-Synergy HT Microplate Reader, Bio-Tek Instruments). Chemotactic index (CI) was calculated from the cell migration activity towards chemoattractant divided by the migration activity in the absence of chemoattractant. The CI values, shown as mean ± SEM, were calculated from five independent experiments.

We measured the activity of GPx with the Glutathione Peroxidase Cellular Activity Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol using lysate obtained as described in 2.8. This kit applies an indirect determination method. It is based on the oxidation of GSH to GSSG catalyzed by GPx, which is then coupled to the recycling of GSSG back to GSH utilizing glutathione reductase and NADPH. We measured the decrease in NADPH absorbance at 340 nm with the Bio-Tek Synergy HT Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA), during the oxidation of NADPH to NADP+ acting as indicative of GPx activity, since GPx was the rate limiting factor of the coupled reactions.

One colony of S. aureus from a tryptic soy agar plate was used to inoculate a 15‐ml sample of Roswell Park Memorial Institute (RPMI; Corning) supplemented with 10 g/L casamino acids (MilliporeSigma) in a 50‐ml conical tube. Samples were supplemented with either 15‐µl DMSO, 10‐µg/ml diflunisal (solubilized in 15‐μl DMSO), Blank‐NPs, or Dif‐NPs (at a final concentration of 10‐μg/ml diflunisal). Samples were prepared in triplicate. Bacteria were grown for 15 h at 37°C and 180 rpm. The triplicate cultures of each group were combined into a single culture of approximately 45‐ml volume and concentrated in Amicon Ultra 3‐kDa nominal molecular weight columns as done previously.⁹, ¹¹ Resulting samples were filter‐sterilized and frozen at −80°C.
To measure the effect of diflunisal and nanoparticles on bacterial growth, 15‐ml cultures were supplemented with DMSO, 10μg/ml diflunisal, PBS, or Blank‐NPs. The bacterial cultures were subsequently grown in 200‐μl volumes in round‐bottom, tissue culture‐treated 96‐well plates for 15 h at 37°C. The optical density at 600 nm (OD₆₀₀) was recorded each hour to monitor bacterial growth using a BioTek Synergy HT microplate reader (BioTek Instruments, Inc.). The initial OD₆₀₀ reading was subtracted from each well to serve as a baseline.