To determine the ability to produce AI-2 of PNC033, the AI-2 bioassay was performed as described previously [26 (link),27 (link)], with some modifications. Briefly, after overnight activating and transferring at a ratio of 1:1000 into fresh LB medium, followed by incubation with BA (25, 50, 100 μg/mL) for 4 h at 37 °C in a shaker at 180 rpm, we collected the supernatant of the strains after centrifugation at 4 °C with 12,000 rpm for 15 min. After filtering through 0.22 μm filters, the samples were stored at −20 °C. Vibrio harveyi BB170, which is an AI-2 reporter strain, was diluted at a ratio of 1:5000 in fresh AB medium, and 180 μL of BB170 culture mixed with 20 μL of the previously supernatant was added to an opaque 96-well plate (Corning Costar, Cambridge, MA, USA) and incubated at 30 °C for 4 hours. Reading was performed using a SpectraMax® i3x (Molecular Devices, LLC, Shanghai, China) in luminescence mode. Vibrio harveyi BB170 was used as positive control. The measure of AI-2 activity was performed as described above, but the supernatant was collected from 0 to 8 h.
Opaque 96 well plate
Manufactured by Corning
Sourced in United States
Opaque 96-well plates are a type of laboratory equipment designed for use in various scientific applications. These plates feature wells with an opaque or light-blocking material that prevents light from passing through the wells. The primary function of these plates is to provide a controlled environment for conducting experiments or assays that require light-sensitive samples or reactions.
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Lab products found in correlation
Celltiter glo luminescent cell viability assay, by Promega (4 mentions)
Celltiter glo reagent, by Promega (3 mentions)
Synergy h4 hybrid reader, by Agilent Technologies (2 mentions)
Glomax luminometer, by Promega (2 mentions)
Caspase glo 3 7, by Promega (2 mentions)
Viktor3, by PerkinElmer (2 mentions)
Imagescope, by Leica (2 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
Aperio digital pathology slide scanner, by Leica (2 mentions)
Imagej, by Leica (2 mentions)
Viktor3 luminometer, by PerkinElmer (2 mentions)
N n 3 5 difluorophenacetyl l alanyl s phenylglycine t butyl ester dapt, by Merck Group (2 mentions)
Celltiter glo luminescent assay, by Promega (2 mentions)
Spectramax i3x, by Molecular Devices (1 mentions)
Dabcyl ktsavlq sgfrkm e edans nh2, by GL Biochem (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Synergy h1 hybrid multi mode reader, by Agilent Technologies (1 mentions)
Fluostar omega microplate reader, by BMG Labtech (1 mentions)
Nano glo luciferase, by Promega (1 mentions)
Ac devd amc, by Cayman Chemical (1 mentions)
24 protocols using opaque 96 well plate
1
AI-2 Bioassay for PNC033 Quorum Sensing
2
Fluorescence-Based Protease Activity Assay
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Purified protein was diluted in reaction buffer (20mM Tris 100mM NaCl 1mM DTT pH 7.3) to a final concentration of 100nM in an opaque 96-well plate (Costar) and incubated with or without compound at varying concentrations for 15 minutes with shaking. The reaction was initiated with the addition of 50μM FRET substrate (Dabcyl-KTSAVLQ↓SGFRKM-E(Edans-NH2); GL Biochem) solubilized in reaction buffer. Cleavage of the substrate generates a product with a free Edans group for which fluorescence was monitored at an excitation wavelength of 360nm and emission wavelength of 460nm. Baseline subtraction was performed for every concentration and all experiments were performed in triplicate.
3
Extracellular Vesicle Luminescence Quantification
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Firefly luciferase and Nanoluc expression in EVs, cells and cerebellum collected with Passive Lysis Buffer (Promega), were analyzed with the addition of luciferin (100mg/mL) or furimazine (Nano-Glo® Luciferase, Promega) diluted 1:200 to 1:500 in 1X PBS, respectively. Samples were incubated with the reagent for at least 1 minutes prior to reading on Synergy H1 Hybrid Multi-Mode Reader (BioTek) or FLUOstar Omega Microplate Reader (BMG LABTECH). At least two reads were performed on each sample, and the average values were considered for analysis. For luminescence readings, samples were loaded into white 96-well culture plates (Lumitrac 200) or opaque 96-well plate (Corning). Each sample was loaded in duplicate with a volume of ranging from 20 to 100 μL in each well.
4
Caspase-3 Activity Assay in MB-MB-231 Cells
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Caspase-3 activity was assessed in MB-MB-231 cells utilizing a previously described protocol [62 (link)] with minor modifications. Briefly, cells were seeded in 48-well plates at a density of 20 × 103 cells/well, cultured overnight and treated with compounds at various concentrations for 24 h. Then, the cells were lysed by applying ice-cold lysis buffer. Next, the samples were transferred to an opaque 96-well plate (Corning) with reaction buffer that contained Ac-DEVD-AMC (a caspase3 fluorogenic substrate; Cayman Chemicals, Ann Arbor, USA). Sample fluorescence was continuously recorded at 37 °C for 2 h using a Biotek Synergy H4 Hybrid Reader (λex 350 nm, λem 460 nm). Crude results for maximal velocity were normalized to the protein content using the SRB method and are reported as a caspase-3 activity fold increase compared to the vehicletreated control (0.1% (v/v) DMSO).
5
FRET-based Factor Xa Activity Assay
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In an opaque 96 well plate Corning), 15 nM of ecotin protein from each
species were mixed with 1 μl of FRET peptide and 20 μl of 10x factor Xa
buffer in a total volume of 200 μl. The samples were analyzed in a plate
reader with an excitation wavelength of 355 nm and emission of 530 nm as
follows: the first scan was blanked and then 5 nM of factor Xa was added to
the corresponding wells. Fluorescence was measured every 5 minutes over a
timeframe of 1 h. A schematic of the FRET assay is depicted inFig 4A .
species were mixed with 1 μl of FRET peptide and 20 μl of 10x factor Xa
buffer in a total volume of 200 μl. The samples were analyzed in a plate
reader with an excitation wavelength of 355 nm and emission of 530 nm as
follows: the first scan was blanked and then 5 nM of factor Xa was added to
the corresponding wells. Fluorescence was measured every 5 minutes over a
timeframe of 1 h. A schematic of the FRET assay is depicted in
6
Quantifying DNA Content in Cells
7
Bisphosphonates Induce Apoptosis in 4T1 Cells
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The 4T1 and 4T1-luc2-tdTomato cells were seeded in 24-well plates at a density of 3×103 cells/ml in the culture medium. The cells were cultured overnight, and treated with WG12399C at a concentration of 23.9 µM or WG12592A at a concentration of 23.6 µM for 24, 48 and 72 h. Untreated cells and cells treated with zoledronic acid (24.1 µM) were used as controls. The concentrations of BPs applied in this assay were based on the calibration curves from antiproliferative tests and did not exceed IC30-40. Following incubation under standard conditions, the cells were lysed for 30 min with ice-cold lysis buffer [50 mM HEPES, 150 mM NaCl, 10% saccharose, 5 mM EDTA, 0.1% Triton-X 100, 10 mM dithiothreitol (DTT), pH 7.5] (IIET). Subsequently, 40 µl of each sample was transferred to an opaque, 96-well plate (Corning, Inc.) containing 160 µl of the reaction buffer (10 µM Ac-DEVD-ACC substrate, 20 mM HEPES, 100 mM NaCl, 10% saccharose, 1 mM EDTA, 10 mM DTT, pH 7.5) (IIET). The fluorescence of each sample was continuously recorded for 2 h at 37°C using the BioTek Synergy H4 Hybrid Reader (λex=360 nm, λem=460 nm). In parallel, the SRB assay was performed in order to normalize to the protein content. The results obtained are expressed as mean relative caspase-3/7 activity in comparison to the untreated cells ± standard deviation (SD).
8
Tape Strip-Based SOD Activity Assay
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The SOD assay was adapted from a well plate method. A D-squame® tape strip was perforated with a punch and three small pieces (=5 mm) were lined at the bottom of a well of an opaque 96-well plate (Corning, Costar, Acton, MA). SOD from bovine erythrocytes was used as an external standard, in which case a blank strip was put in the well. SOD activity was assayed using Sigma SOD assay kit (19160, Sigma, USA) following the manufacturer's instructions. The reaction started immediately when 220 μl of reaction mixture was added to each well. This assay is based on the xanthine/ xanthine oxidase catalytic system. After SOD measurement, the tape strips were removed and stored in an Eppendorf tube for protein analysis. Specific SOD activity was obtained after normalizing to the total protein content on the stripping.
9
Rigosertib Sensitivity in PDX Organoids
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LU-NB-1, LU-NB-2, and LU-NB-3 PDX-derived tumor organoids were dissociated into single cells and seeded into opaque 96-well plates (Corning Inc., Corning, NY), 5000 cells per well, and treated immediately with a range (0–100 nM) of rigosertib concentrations. Cells were incubated for 72 h. Cell viability was calculated as a percentage of control wells based on CellTiter-Glo (G7571; Promega, Madison, WI) luminescence. Luminescence was measured with a Synergy2 Multi-Mode plate reader (BioTek, Winooski, VT). Biological triplicates were used.
10
Cell Viability and Caspase Activity
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