Trypsin inhibitory assay was performed in a solution containing 20 mM CaCl2, 50 mM Tris•HCl buffer at pH 8.0. Inhibitor solutions of SFTI-1 ranging from 1 pM nM to 100 nM were preincubated with a freshly prepared solution of 5 nM trypsin in a final reaction volume of 100 μL. After incubation for 30 minutes, residual trypsin activity was measured by adding the fluorogenic substrate Nα-Cbz-L-Arg-AMC (AMC: 7-amido-4-methyl-coumarin, Cbz: benzyloxycarbonyl) to a final concentration of 3 μM and then following the release of AMC using a using an Envision 2103 plate reader (PerkinElmer). Measurements were taken every 30 seconds using an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The initial velocities for the hydrolysis of substrate Nα-Cbz-L-Arg-AMC by trypsin in the presence of different concentrations of SFTI-1 were fitted to a one-site competitive binding equation using the software package Prism (GraphPad Software). Ki was calculated using the equation of Cheung and Prusoff [38 (link)] and a Km value of 12 μM [39 ].
Envision 2103
Manufactured by PerkinElmer
Sourced in United States
The Envision 2103 is a multimode microplate reader designed for high-performance detection of a wide range of assays. It features a flexible optical system with up to six detection modes, including absorbance, fluorescence intensity, time-resolved fluorescence, luminescence, and AlphaScreen®.
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Lab products found in correlation
Munana, by Merck Group (3 mentions)
Celltiter glo cell viability assay, by Promega (3 mentions)
Munana, by PerkinElmer (2 mentions)
Prism 7, by GraphPad (2 mentions)
Celltiter 96 aqueous one solution, by Promega (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Black 384 well plate, by Greiner (2 mentions)
Nunc immuno plate, by Thermo Fisher Scientific (1 mentions)
Huab25 hrpo, by Johnson & Johnson (1 mentions)
Alphalisa detection kit, by PerkinElmer (1 mentions)
Bio gel p 4, by Bio-Rad (1 mentions)
Celltiter glo 2, by Promega (1 mentions)
Flexi rabbit reticulocyte lysate system, by Promega (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Delfia cell proliferation kit, by PerkinElmer (1 mentions)
S1060, by Selleck Chemicals (1 mentions)
S8007, by Selleck Chemicals (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
Celltiter 96, by Promega (1 mentions)
Cyquant kit, by Thermo Fisher Scientific (1 mentions)
48 protocols using envision 2103
1
Trypsin Inhibition Assay for SFTI-1
2
Leukocidin Promoter Activity in S. aureus
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To measure leukocidin promoter activity in S. aureus during DC interactions, bacteria containing reporter plasmids (individual leukocidin promoters fused to the luciferase operon from Photorhabdus luminescens present in the pXEN plasmid [Xenogen]) (11 (link)) were used. For each experiment, bacteria were freshly streaked onto TSA plates supplemented with 10 μg/ml chloramphenicol and bacteria were grown overnight in 5 ml tryptic soy broth (TSB; BD Difco) supplemented with 10 μg/ml chloramphenicol at 37°C under shaking conditions (180 rpm). The following day, bacteria were first subcultured 1:100 in fresh RPMI medium–Cas for 3 h and subsequently subcultured 1:10 for another 3 h in fresh RPMI medium–Cas to reduce background luminescence. Cells and bacteria were coincubated at an MOI of 10 at 37°C under shaking conditions at 180 rpm. Luminescence readings were taken at time points from time point zero to 4 h using a PerkinElmer Envision 2103 multilabel reader. Values corresponding to the background fluorescence of medium alone were subtracted from the readings determined for the samples.
3
Influenza virus neuraminidase activity assay
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2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA, Sigma, M8639) is a fluorescent substrate for the neuraminidase (NA) of influenza viruses and is used to detect the replication levels of influenza viruses; therefore, it can also be used to measure the inhibitory effect of agents against influenza virus infection (An et al.2014 (link)). Briefly, the virus-containing culture supernatant was transferred to a black opaque 96 or 384 well plate (PerkinElmer, 6005270 or 6007270) and mixed with 20 μmol/L of MUNANA dissolved in MES solution (33 mmol/L 2-[N-morpholino] ethanesulfonic acid and 4 mmol/L CaCl2, pH = 6.5), followed by incubation at 37 °C for 1 h. The reaction was terminated by the addition of stop solution (0.14 mol/L NaOH in 83% ethanol). Fluorescence intensity was measured at an excitation wavelength of 355 nm and an emission wavelength of 485 nm using multi-label plate readers (Envision2103, PerkinElmer, USA).
4
Quantification of Amyloid-Beta in ELISA
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For detection of human and mouse Aβ, an in-house ELISA sandwich was carried out. Briefly, 96-wells Nunc-Immuno plates (Nunc, Denmark) were coated overnight at 4 °C with JRF AB038 antibody for Aβ1‐38, JRF cAb040/28 antibody for Aβ40 or JRF Ab042/26 antibody for Aß42 (Janssen Pharmaceutica), all used at 1.5 mg/ml in PBS containing 0.1% casein (Casein Buffer). Plates were washed 5 times with Washing Buffer (PBS-0.05% Tween 20) before the addition of the samples or the standard curve made with consecutive dilutions (from 100 to 0.0003 ng/ml) of human or mouse Aβ40 and Aβ42 (rPeptide). Detection antibody was obtained from Janssen; huAB25‐HRPO. After overnight incubation at 4 °C and 5 time washes with the Washing Buffer, the samples were developed with a 0.02% TMB (tetramethylbenzidine) solution in Sodium Acetate (100 mM pH 4.9) containing 0.03% H2O2. The reaction was stopped with 0.2 N H2SO4 and read at 450 nm on a Perkin Elmer Envision 2103 multilabel reader.
5
Cell Line Viability Assay for Vandetanib
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Cell line IC s for vandetanib were obtained for a panel of 20 cell lines. Cells were plated on 96-well plates at a concentration of 15,000 cells/mL (3000 cells/well) prior to treatment with vandetanib (serially diluted from 160 M to 0.25 M) for 24 h. IC was measured five days following drug treatment via cellular metabolism of resazurin. Cell culture media was replaced with FluorobriteTM media containing 10 g/mL resazurin. Resazurin metabolism into fluorescent resorufin was monitored for up to 4 h, at which point fluorescence emission at 590 nm was measured with a Perkin Elmer Envision 2103 plate reader (Waltham, MA, USA), and the dose corresponding to 50% survival was determined from the survival curves using Graphpad Prism (San Diego, CA, USA).
6
Insulin Secretion Dynamics in Pancreatic Islets
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After overnight incubation, approximately 80 islets from each pancreas were transferred to a chromatograph column (PERI-4.2, BioRep technologies, Miami Lakes, FL, USA) filled with Bio-Gel P-4 (Bio-Rad Laboratories, Hercules, CA, USA) to stabilize them during the perifusion. Islets were pre-perifused with NaCl (125 mM), KCl (5.9 mM), CaCl2 (1.28 mM), MgCl2 (1.2 mM), HEPES (25 mM), BSA (0.1%) and glucose (3 mM), pH 7.4 for 45 min at 37 °C. The islets were perifused in the buffer above for 12 min, then sequentially exposed to 11 mM glucose for 35 min followed by 3 mM glucose for 15 min and the protocol finished with 25 mM KCl + 3 mM glucose for 15 min to empty all possible insulin granules. Fractions (50 μl) of the perifusate were collected every min during stimulation in a 96-well plate. The collected fractions were then measured for insulin concentration by the AlphaLISA detection kit (PerkinElmer, Waltham, MA, USA) with a plate reader (EnVision2103, PerkinElmer, Waltham, MA, USA).
7
Neuraminidase Activity Assay for Influenza Viruses
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The fluorescent substrate, 2′-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA, Sigma, M8639), for the neuraminidase (NA) of influenza viruses was used to detect the levels of NA. Briefly, the virus-containing culture supernatant was transferred to a black opaque 96 or 384 well plate (PerkinElmer, 6005270 or 6007270) and mixed with 20 µmol/L of MUNANA dissolved in MES solution (33 mmol/L 2-[N-morpholino] ethanesulfonic acid and 4 mmol/L CaCl2, pH = 6.5), followed by incubation at 37 °C for 1 h. The reaction was terminated by the addition of stop solution (0.14 mol/L NaOH in 83% ethanol). Fluorescence intensity was measured at an excitation wavelength of 355 nm and an emission wavelength of 485 nm using multi-label plate readers (Envision2103, PerkinElmer, Waltham, MA, USA).
8
Cell Viability Assay Protocol
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To determine viability, cells were seeded to opaque white 96-well plates. The next day, cells were drug- or vehicle-treated and incubated as in infection experiments (for example, treated-MRC-5 cells were placed for 48 h at 33°C). ATP levels were then assayed using Celltiterglo2.0 (Promega G9242) according to the manufacturer's instructions. Luciferase signal was read on a Perkin Elmer Envision 2103 multilabel reader, and 24-h 1 µM staurosporine treatment used as a positive control for assay sensitivity.
9
In vitro Firefly Luciferase Translation
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In vitro translation of firefly luciferase with WT and mutated F-IRES mRNA (1 μg in 50 μL of reaction mixture) was performed using the Flexi Rabbit Reticulocyte Lysate System (Promega) according to the manufacturer. IRES mRNA was omitted in the control reaction. Fluc activities (Figure 1—figure supplement 1 ) were determined using a plate reader (Envision 2103, Perkin-Elmer) to detect the luminescence signal.
10
Quantitative Dye Analysis of CaCO3
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Quantitative analysis was conducted of EDTA-solubilized samples of known mass against a calibration curve of known [Dye] in 96-well plates (Greiner Black μClear) to facilitate mol% dye versus CaCO3 calculations. Plates were analysed with a Perkin Elmer EnVision 2103.
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