Victor x

P-glycoprotein activity was measured using the calcein-AM assay. CHO-APP and SK-N-SH cells were seeded into 96-well clear-bottom black-walled plates at 4 × 10⁴ cells/well in full culture medium and incubated overnight. The next day when cells reached ~80–90% confluency, media was discarded, and the cells were washed with phenol red-free HBSS or MEM. To assess the inhibitory effect of verapamil and nicardipine on P-gp activity, the inhibitors and DMSO control were prepared at 2× concentrations in HBSS buffer and added to the cells at 100 µL/well. Calcein-AM substrate was also prepared at 2× concentration, and 100 µL was added to each well using a multi-channel pipette to achieve the final working concentrations. Fluorescence measurements were obtained every minute for 20 min, starting immediately after addition of calcein-AM, using the Perkin Elmer Victor X plate reader. The excitation and emission wavelengths were set at 485 and 535 nm, respectively. Temperature was maintained at 37 °C. Measurements were recorded in relative fluorescence units (RFU) and computed using Graphpad Prism software.

MIN-6 proliferation was evaluated at day 10 after seeding by the MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega Corporation, Madison, WI, USA). After removing the culture medium, the cells were washed with PBS (pH 7.4). After this, 1000 μL serum-free DMEM medium and 200 μL MTS solution were added to sample wells and incubated for 1.5 h at 37 °C and 5% CO₂ atmosphere. Obtained reaction products were collected and put into 96-well plates (150 μL per well). The optical density of the plate wells was measured using a microplate reader (Victor X, Perkin Elmer, Waltham, MA, USA) at 490 nm. Cells were washed with PBS (3 × 5 min) and cultured further with regular medium.

The extent of biofilm formation by the various C. albicans strains was measured by a 96-well microtiter plate biofilm assay [40 (link)]. Briefly, the overnight grown cells were washed with PBS and then diluted to 10⁷ cells/ml, in various media, and 100 μl of each suspension was transferred in quadruplicate into 96-well polystyrene, round-bottom microtiter plates (Thermo Fisher Scientific). Medium without C. albicans cells was used as a baseline control. The extent of biofilm was quantified after 24 h by crystal violet staining. First, the wells were washed twice with 200 μl of PBS and then dried for 45 minutes at room temperature. Then, 110 μl of 1% crystal violet solution (Thermo Fisher Scientific) was added and incubated for 45 minutes at room temperature. The wells were then washed four times with 350 μl sterile water to remove the unbound crystal violet. Finally, the biofilm-associated crystal violet was solubilized with 110 μl of 95% ethanol (Sigma-Aldrich) at room temperature for 45 min. Subsequently, 100 μl was transferred into a new 96 well plate for reading at 570 nm (VictorX, Perkin Elmer, Waltham, MA).

Subcellular fractionation was conducted as described above. In cases were cells have been treated with substances, all buffers used during the fractionation have been supplemented with these substances accordingly. Purified mitochondria were resuspended in KCl buffer supplemented with protease inhibitors and indicated substances and aliquoted to one sample per time point for western blot analysis and two samples per time point for fluorometric measurements. After incubation at 37 °C for the indicated time, each sample was put on ice shortly and separated into mitochondrial pellet and supernatant by centrifugation for 5 min at 10,000 × g at 4 °C. For western blot analysis mitochondrial pellets were washed once with KCl buffer while supernatant samples were acetone precipitated. The resulting pellets of both fractions were boiled in SDS sample buffer and subjected to SDS-PAGE and western blot.
GFP-tagged BAX was measured in duplicates by fluorometry, using a PerkinElmer Victor X plate reader with excitation filters at 485 nm and emission filters at 535 nm wavelengths. Mitochondrial pellets were resuspended in KCl-buffer containing 1% (v/v) Triton X100 and centrifuged for 5 min at 10,000 × g at 4 °C prior measurement.

To estimate the release profile of Rho B from the HMP-DMN patch, phosphate-buffered saline (PBS, pH 7.4; Life Technologies, Carlsbad, CA, USA) containing 25% (v/v) ethanol was used as the releasing medium [29 (link)]. The HMP-DMN patch was fabricated, and the amount of Rho B encapsulated in the 3 × 3 arrays of the HMP-DMN was calculated using the following formula:
The control DMN patches were fabricated using 60% (w/v) HA solution containing Rho B by carrying out a round of centrifugation at 3470× g for 2 min without PLGA. The amount of Rho B in the control DMN patch was the same as that in the HMP-DMN patch. To analyze the release of Rho B, control DMN patches and HMP-DMN patches were placed in 5 mL of PBS in a conical tube and then placed on a hot plate stirrer at 37 °C and 100 rpm for 26 days. Every 2 days, the released PBS was replaced with 5 mL of fresh and preheated medium. The replaced PBS was analyzed using a multimode plate reader (VICTOR™ X, PerkinElmer, Waltham, MA, USA) to estimate the amount of Rho B in each patch.

Two hundred μl of autoinducer bioassay (AB) mineral medium (0.3 M NaCl, 0.05 M MgSO₄, 0.5% casein hydrolysate, 10 μM KH₂PO₄, 1 μM L-arginine, 50% glycerol, 0.01 μg/ml riboflavin, 1 μg/ml thiamine. pH 7. Sigma-Aldrich) containing 10% (V/V) of a tenfold dilution of an overnight culture of Vibrio harveyi BB170 (ATCC BAA-1117) grown in AB medium were supplemented with 10 mg/l of E. acoroides leaf extract respectively, and were placed in hydrophobic 96-well polystyrene-based microtiter plates (Thermo Fisher Scientific) with transparent bottom. The positive control was an AB mineral medium supplemented with 10% (V/V) tenfold dilution of the overnight culture. Absorbance (OD_600nm) and luminescence were measured using a microplate reader (VICTOR™X, Perkin Elmer, USA) every 8 h for 24 h, incubating the microtiter plate at 30 °C during the experiment. The data obtained were normalized to the number of viable cells, divided by the area of the membrane, and the means reported. The experiment was repeated three times.