Victor multilabel plate reader

PC3 and VCaP cell lines were purchased from ATCC. Cytotoxicity of different test compounds were studied on both PC3 and VCaP cell lines by determining the number of viable cells based on quantitation of ATP using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Mannheim, Germany).
Cells were seeded into 384-well plates (Corning Life Sciences, Tewksbury, MA, USA) at 1000 cells/well density in 40 μl media and incubated for 4 h at 37°C. Test compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma, Budapest, Hungary) and cells were treated with an increasing concentration of drugs (1.11–90 μM). The highest applied DMSO content of the treated cells was 0.5%. After 48-h incubation at 37°C under 5% CO₂, 40 μl CellTiter-Glo® Reagent (Promega) was added to each well and the luminescent signal was recorded by luminometer (VICTOR Multilabel Plate Reader, Perkin Elmer). Viability was calculated with relation to untreated control cells after extracting signals from blank wells containing only culture medium. IC50 values (50% inhibiting concentration) were calculated by GraphPad Prism®5 (La Jolla, CA, USA).

MHCC97L cells were cultured in soft and stiff matrix gels for 7 days. Histones were extracted using an EpiQuik Total Histone Extraction Kit (EpiGentek). Global histone H3 modification in the soft matrix environment was determined using an EpiQuik Histone H3 Modification Multiplex Assay Kit (EpiGentek). The results were read on a VICTOR multilabel plate reader (Perkin Elmer) at 450 nm and normalized based on the total H3 levels.

Green fluorescent protein (GFP) and luciferase-expressing tumor cells (GFP/Luc⁺) were counted and resuspended at a concentration of 3 × 10⁵ cells/mL. Tumor cells were given D-Luciferin at 75 µg/mL final concentration (Perkin Elmer) and were placed in 96-well white flat-bottomed plates at 100 µL/well, in triplicate. CAR T cells were added to the wells at 10:1 E:T ratio. Target cells incubated without CAR T cells or in the presence of 1% Triton™ X-100 (Sigma-Aldrich) were used to detect spontaneous and maximal killing, respectively. Cells were incubated at 37 °C and the bioluminescence (BLI) was measured as relative light units (RLU) at indicated timepoints with a luminometer (VICTOR Multilabel Plate Reader, Perkin Elmer) and WorkOut v2.5 software (Perkin Elmer). Lysis percentage was calculated using the following equation: % specific lysis = 100 x (spontaneous cell death RLU – sample RLU) / (spontaneous death RLU – maximal killing RLU).

LAT was analyzed by high-performance liquid chromatography (HPLC).
The assay was performed in a Waters 2695 HPLC equipped with a Waters
2996 detector (Waters Corporation, Milford, MA). Briefly, a mobile
phase (acetonitrile:water, 60:40 v/v) flowed isocratically through
a Xterra MS (reference 186000494) C18 HPLC column (250 × 4.6
mm, 5 μm) (Waters Corporation, Milford, MA) at a flow rate of
1 mL/min. The injection volume was 20 μL, and the wavelength
was 210 nm. The sample and column were maintained at 25 °C. The
limit of quantification (LOQ) of the analytical technique was set
at 0.1 ng/mL.
Fluorescein was quantified with a UV spectrophotometer
(VICTOR Multilabel Plate Reader, PerkinElmer, Waltham, MA) at the
wavelength of 485 nm.
Encapsulation efficiency, according to
the indirect method, was
calculated according to eq 1. where W_NE is the
amount of drug quantified in the filtrate (drug not encapsulated)
and W_T the drug quantified in the total
formula. Liposomes were centrifuged in 100 kDa Amicon Ultra units
(Merck KGaA, Darmstadt, Germany) at 4500 rpm for 30 min.

Collagen films were washed once with PBS and pre-conditioned with cell culture media for 1 h before cell seeding. Cardiomyocytes were dissociated using TrypLE (Life technologies) and plated at 10⁵ cells per film in CDM BSA supplemented with ROCK inhibitor 1 μM. For co-cultures, endothelial cells were coated alongside at a ratio of endothelial cells to cardiomyocyte of 1:10 in STEMPRO-34 supplemented with VEGF-A (50 ng/ml). PrestoBlue® Cell Viability Reagent (Thermo Scientific) was added to culture media according to the manufacturer's instructions. Cells were incubated with the dye for 2 h. Media was then sampled and fluorescence at 560 nm analysed using VICTOR Multilabel Plate Reader (PerkinElmer). Media containing PrestoBlue® incubated in empty wells was used as background control.

A Prestoblue^® working solution was prepared in a growth culture medium without phenol red according to the manufacturer’s instructions. Briefly, the culture medium was removed from cell culture wells and a Prestoblue working solution was added and incubated at 37 °C, 5% CO₂. After 3 h, the well volumes were collected in a new 96-well plate and the absorbance was read at λ = 570 nm (experimental) and λ = 600 nm (reference wavelength for normalization) using a Victor Multilabel plate-reader (Perkin-Elmer, Wellesley USA).

The adherence of the L. amylovorus strains to porcine gastric mucins (type II, Sigma) or to porcine small intestinal mucus was studied essentially as described earlier [35 (link)], but by using a nucleic acid binding fluorescent stain SYTO^®9 (Molecular Probes, Eugene, OR) rather than tritium for bacterial labeling, and PBS as the buffer. To label the bacterial cells in the experiments, the strains were cultivated overnight, collected, washed twice with 0.85% NaCl and suspended into the original volume of 0.85% NaCl, and then 1 μl of 5 mM SYTO^®9 solution was added per 1 ml of cell suspension, followed by a 15 minute incubation in the dark with vigorous shaking, after which the cells were collected and washed twice with PBS. After the adherence assay, the input (added) and output (remaining) fluorescence values were measured in a microplate reader (Victor Multilabel Plate Reader, Perkin Elmer, Waltham, MA) and the adherence was expressed as the proportion (%) of the original fluorescence remaining, after first subtracting the background fluorescence from mucus-coated wells without bacteria (for outputs) and from wells filled with PBS (for inputs).