Chameleon 5 plate reader

The production of intracellular reactive oxygen species (ROS) was measured in Huh7-luc/neo cells after 24 h of incubation with compounds. Then, medium was removed, and cells were incubated with 10 µM 2′,7′-dichlorofluorescein diacetate (DCF-DA) and washed 10 times with 500 µl of PBS. The fluorescence intensity (DCF intensity) was measured in 200 µl PBS using a Plate CHAMELEON V reader (Hidex, Turku, Finland) with excitation at 485 nm and emission at 535 nm.

Intracellular reactive oxygen species (ROS) production was measured by epifluorescence using the described protocol [26 (link)]. Briefly, the Huh7 cells were seeded into 48-well plates, transfected with plasmids encoding HCV core variants. Cell culture medium was removed 30 h post transfection, and cells were incubated in the medium containing 25 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min. The treated cells were washed with PBS, resuspended in PBS, and the fluorescence intensities were measured using Plate CHAMELEON V reader (Hidex Oy, Åbo, Finland) with excitation at 485 and emission at 535 nm.

Caco-2^nfkb-RE [9 (link)] cells were seeded in 96-well plates (Corning Incorporated, New York, NY, USA). At confluency, inhibitors of endocytosis were added to the cells and incubated as indicated below. Subsequently, cells were treated with TiO₂ NPs or water (control) at a final concentration of 40 µg cm⁻² cell growth surface. After 6 h cells were harvested and luciferase activity was measured for 5 s in a CHAMELEON™ V plate reader (Hidex, Finland) using Beetle-Juice substrate (PJK GmbH, Kleinblittersdorf, Germany) according to the manufacturer’s instructions. Luciferase activity is expressed in relation to untreated controls.

Briefly, 1 × 10⁴ cells were plated into 96-well plate for 24 h and serum-starved for additional 24 h, before treated with complete media, control media (media containing 1% FBS) or fibroblast conditioned media (1.0 μg/μl) with or without chemicals, recombinant proteins or neutralizing antibodies (BioLegend, CA, USA) for subsequent 24-72 h. At the end of treatment, MTT solution (5 mg/ml) was added into each well, followed by 4 h incubation at 37°C. The formazan crystals were dissolved with sodium dodecyl sulfate before reading the absorbance using CHAMELEON™V plate reader (Hidex, Finland) at 570 nm with reference wavelength of 630 nm.
To measure cell proliferation, we determined BrdU incorporation into the cellular DNA using an ELISA-based approach (Cell Signaling Technology, MA, USA). Cells were seeded and treated as described above, and at the end of treatment, BrdU solution was added for 24 h. Following fixation and denaturation, the cells were labeled with antibodies and washed before added with TMB substrate. Color development was terminated with a STOP solution, and measured at 450 nm using CHAMELEON™V plate reader. In addition, the cells were also stained with 0.04% trypan blue to count for number of viable cells, using neubauer improved hemocytometer (Sigma Aldrich).

Methods were adapted from Martins-Green et al. [55 (link)]. Transwell inserts were coated with growth factor-reduced Matrigel^® (Corning) and seeded with HUVECs and HLMVECs in Vasculife-VEGF or Vasculife-MV media (1 × 10⁵ cells/well). A second monolayer was seeded 48 h later. The assay was initiated 48 h later with the addition of 10 μg/mL of the final concentration of FITC-dextran sulfate (MilliporeSigma) and 100 ng/mL of the EVs to the lower chambers, while inhibitors (100 μM EA, MRS2179, or both) were added to top chambers. Media was collected from the top chambers at indicated time points and fluorescence was measured with 485 nm excitation/535 nm emission using the Hidex Chameleon V plate reader and MikroWin 2000 software version 4.43 (Hidex). n = 6.

RAW-ELAM (35 (link)) cells (1 × 10⁵ cells/well) in phenol red-free DMEM were added to white 96-well plates and incubated for 12 to 16 h. Medium was removed, and purified iRBCs or uRBCs (5 × 10⁵ cells/well) in 100 μl medium were added. Plates were incubated for a further 12 h. BriteLite Plus luciferase reagent (100 μl; PerkinElmer) was added to wells, and the wells were incubated for 3 min. Total luminescence was measured on a Chameleon V plate reader (Hidex). Samples were analyzed in quadruplicate, and the vehicle negative control and the Ultrapure LPS-EB (lipopolysaccharide from E. coli 0111:B4; InvivoGen) positive control were included. Values for uRBC samples were subtracted from those for iRBC samples. Data were normalized to represent fold changes of PfEMP1-null iRBC stimulation over WT iRBC stimulation and of LPS stimulation over PBS stimulation, where the value for the WT was equal to “1,” and the luciferase levels measured from PfEMP1-null cells were determined relative to the WT signal. Independent experiments were repeated a minimum of three times on separate days and with freshly isolated iRBCs.

Cellular toxicity caused by hsp90 inhibition was measured using a luminescent cell viability assay based on quantitation of ATP (ToxGlo; Promega). Cells were grown in μclear 96-well plates (Greiner Bio-One) and maintained in phenol red-free DMEM (Life Technologies). A dilution series of drug inhibitor was made in phenol red-free medium, and 100 μl replaced the medium in each well. For antiviral dose-response experiments, FMDV was added at an MOI of 0.01. At the end of the toxicity period required, room temperature ATP detection reagent was added to ATP detection substrate and mixed thoroughly, and 100 μl was added to each well. After 10 min, luminescence was detected using a Chameleon V plate reader (Hidex).