The time-dependent consumption of thiols in each hydrogel formulation was quantified via modified Ellman’s assay. Briefly, 5 µL of the hydrogel mixture was incubated for 2 h at 37 °C. The gel disk was then mixed with 250 µL of Ellman’s Reagent (50 mM sodium acetate and 10 mM DTNB in DI water) and 100 µL of PBS. Samples were rotated at 1,400 RPM on an Eppendorf thermomixer (Hamburg, Germany) at 50 °C for 10 min. Subsequently, 645 µL of DI water was added and the mixture was vortexed at 50 °C for 5 min. The solution absorbance was recorded at 405 nm on a Wallac 1420 Victor2™ plate reader (Perkin Elmer, Rodgau, Germany). The time-dependent thiol consumption was calculated by comparison to a cysteine standard. For time points beyond 2 h, the hydrogel was allowed to swell with 40 µL of PBS and at the time of analysis the PBS volume was made up to 100 µL.
Wallac 1420 victor2 plate reader
Manufactured by PerkinElmer
Sourced in United States
The Wallac 1420 VICTOR2 plate reader is a multi-mode microplate reader designed for a variety of applications in life science research. It is capable of absorbance, fluorescence, and luminescence measurements. The device is equipped with a temperature-controlled incubator and shaker for precise control of experimental conditions.
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Lab products found in correlation
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (2 mentions)
Thermomixer, by Eppendorf (1 mentions)
Nunc 96 well polisorp plates, by Thermo Fisher Scientific (1 mentions)
Delfia enhancement buffer, by PerkinElmer (1 mentions)
Delfia assay buffer, by PerkinElmer (1 mentions)
Celltiter96 non radioactive proliferation assay, by Promega (1 mentions)
Compusyn software, by ComboSyn (1 mentions)
Lucigenin, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Lonza (1 mentions)
Celltiter glo reagent, by Promega (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Black 96 well microplates, by PerkinElmer (1 mentions)
Coelenterazine h, by Promega (1 mentions)
Celltiter 96 aqueous one solution reagent, by Promega (1 mentions)
96 well plate, by Corning (1 mentions)
14 protocols using wallac 1420 victor2 plate reader
1
Quantifying Time-Dependent Thiol Consumption in Hydrogels
2
Cell Viability Assay Protocol
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Cell viability was assessed by CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. Each treatment within a single experiment was performed in triplicate. Absorbance at 490 nm was recorded by a Wallac 1420 VICTOR2 plate reader (PerkinElmer, Waltham, MA, USA). Data were normalized to untreated control.
3
Quantitative Glycan Profiling Assay
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Gradient fractions were diluted in 0.5 M GuHCl and coated on to Nunc 96-well polisorp plates (ThermoFisher Scientific, 475094) overnight at 4°C. The plates were washed 3 times with DELFIA washing solution (5 mM Tris-HCl, 0.15 M NaCl, 0.005% Tween 20, 0.01% NaN3, pH 7.75). Periodate-oxidation was performed for 20 min with 25 mM sodium metaperiodate in 0.1 M sodium acetate buffer (pH 5.5). The plates were washed again 3 times with DELFIA washing solution and one time with PBS/0.05% Tween, and the wells were blocked for 1h with DELFIA blocking solution (50 mM Tris-HCl, 0.15 M NaCl, 90 µM CaCl2, 4 µM EDTA, 0.02% NaN3, and 0.1% BSA) at 22–24°C. After discarding the blocking buffer, the plates were incubated for 1 hour with 2.5 µM biotin-hydrazide solution in 0.1 M sodium acetate buffer (pH 5.5) at 22–24°C. The plates were washed again 3 times and then incubated for 1 hour at 22–24°C with europium labelled Streptavidin (PerkinElmer, 1244–360) 1:1,000 in DELFIA assay buffer (PerkinElmer, 1244-111). This was followed by washing the plates six times with DELFIA washing solution and incubated for 5 min on a shaker with DELFIA enhancement buffer (PerkinElmer, 1244-114). The fluorescence was measured using a Wallac 1420 VICTOR2 plate reader with Europium label protocol (PerkinElmer, Waltham, MA, USA).
4
Proliferation of Embedded Cells in Fibrin Gel
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Cells (ASCs or fibroblasts: 3 × 105) were embedded in the fibrin gel (100 μL) and cultivated in culture medium for 24 hours before medium was discarded and proliferation of cells was measured using a CellTiter96 Non-Radioactive Proliferation Assay (Promega Corporation, Madison, WI). Fibrin clots with no cells were used as negative control. Cell number was evaluated according to manufacturer's protocol: supernatants were discarded and 45 μL dye solution was added to 300 μL culture medium. After 1.5 hours of incubation at 37°C in a humidified atmosphere with 5% CO2 stop solution was added. After one hour of incubation 100 μL of the supernatants was transferred in triplicates on a 96-well plate and absorbance was measured at 555 nm on a Wallac 1420 VICTOR2 plate reader (PerkinElmer, Waltham, MA, USA). Further proliferation assays were performed on day 7 and day 14 of the experiment. Additionally, fibrin clots with no cells were used for negative control measurement. Fibrin clots with cells from at least 4 different donors were analyzed in independent experiments.
5
Evaluating Synergistic Drug Interactions
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Cell viability was assessed by CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer’s protocol. Each treatment within a single experiment was performed in triplicate. Absorbance at 490 nm was recorded by a Wallac 1420 VICTOR2 plate reader (PerkinElmer, Waltham, MA, USA). Data were normalized to untreated control. The nature of the interactions between 5-azanucleosides and topoisomerase inhibitors was analyzed by the Chou-Talalay method [18 (link), 19 (link)] using CompuSyn software (ComboSyn, Paramus, NJ, USA). Combination Index (CI) values for each drug combination were determined as a quantitative measure of drug-drug interaction. A CI value of < 0.9 indicates synergy, a CI value of 0.9–1.1 indicates additive effect, and a CI value of > 1.1 indicates antagonism.
6
Quantifying Cell Viability and Proliferation
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Cells were seeded in white 384 well plates in at a pre-determined optimal seeding density. After 24 hr cells were treated with a 10-fold serial dilution of GSK126 or 0.1% DMSO (vehicle control). Following 3 days of incubation cells were lysed and viability was measured using CellTiter-Glo (CTG, Promega) luminescence detection using a Perkin Elmer Wallac 1420 VICTOR2 plate reader. CTG values were normalized to a percentage of control cells and plotted as a dose-response curve. The concentration of GSK126 required to inhibit 50% of cell growth (IC50) was determined. For proliferation assays an IncuCyte 2011A device captured images of cells growing in a 96-well plate at regular intervals and mean confluence was calculated. Untreated cells were imaged to determine the starting number of cells at time zero (T0) and data expressed relative to T0 in response to drug treatment over time.
7
BoNT-Induced Changes in Cell Proliferation
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Keratinocytes and HUVECs were seeded in 96-well plates (8 × 104 cells per well). After 24 hours of cultivation, medium was changed to BoNT-containing proliferation medium. As a control, normal proliferation medium without BoNT was added to the cells. The cell number on days 1, 2, and 3 was evaluated using a CellTiter96 nonradioactive proliferation assay (MTT-Assay, Promega; Madison, Wis.) according to the manufacturer’s protocol, and absorbance was measured on a Wallac 1420 VICTOR2 plate reader (PerkinElmer; Waltham, Mass.).
8
Quantifying Neutralizing Antibodies in Hemophilia B
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Human sera samples were collected from 10 hemophilia B patients at the Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical Sciences and Pekin Union Medical College (Tianjin, China), which was approved by the ethics review board. Sera samples from rhesus monkeys were obtained from Greentech Bioscience (Sichuan, China). NAb levels were detected following our previous protocols, with minor modifications.42 (link),70 (link) Briefly, the rationale of the ratio to mix AAV and sera (or sera dilutions) was grossly based on an intravenous administration at a dose of 1 × 1013 vg/kg, namely 2 × 108 vg vps per 1 μL serum. Huh7 cells were cultured at the density of 1 × 105 cells/well in a 48-well plate. A serial 2-fold dilution of sera samples of 5 μL in volume each were incubated with 1 × 109 vg AAV particles for 2 h at 4°C, followed by treatment of the Huh7 cells with the AAV-sera mixture. The activity of luciferase delivered by AAV was measured by the Wallac-1420 Victor 2 plate reader (PerkinElmer, Waltham, MA, USA) 48 h post-AAV treatment. NAb titers were defined as the largest number of dilution ratios able to reduce the luciferase activity by half compared with the AAV-PBS mixture group. A serum sample was considered NAb positive when the titer was higher than 1:4.
9
Cell Viability Evaluation Using CellTiter96
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Viability of cells was further measured using a CellTiter96 non-radioactive proliferation Assay (Promega Corporation, Madison, WI). Therefore, cells were seeded on fleeces in 24 well cell crowns (6×104 cells). On day one, three and seven cell viability was evaluated according to manufactureŕs protocol. Therefore, 15 µl dye solution was added to 100 µl medium, after two hours of incubation 100 µl stop solution was added and incubated for one hour in the dark. Fleeces were gently shaken to homogenize medium and substrate, 50 µl of the supernatant was transferred into a 96 well plate in triplicates and absorbance was measured on a Wallac 1420 VICTOR2 plate reader (PerkinElmer, Waltham, MA, USA).
10
Quantifying Superoxide Anion via LECL
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For O2·- quantification via LECL 100 μg protein was added to each well in a 96-well plate, solid white with a flat and transparent bottom (Greiner Bio-One™). Briefly, red phenol-free Hank's Balanced Salt Solution (HBSS, Lonza) supplemented with HEPES (1 mM) pH 7.4 was mixed in a 1:1 v/v ratio with Na2CO3/NaHCO3 0.1 M (4:1 v/v) buffer, pH 11.0; final pH was adjusted with NaOH or HCl 1 M to 10.4. In some cases, protein lysates were incubated with pharmacological inhibitors, febuxostat (1 μM) or DPI (10 μM) for 30 min at 37 °C. Experimental substrate(s) xanthine, NADPH or NADH were added at 100 μM per well, followed by 10 μM lucigenin (Sigma Aldrich, UK) as previous studies [37 (link)]. Luminescence was immediately analysed using a PerkinElmer Wallac 1420 Victor 2 plate reader (PerkinElmer, USA), O2·- anion production was read for 50 repeat readings with 30 s interval between each repeat. Each sample was run in duplicate, and the results averaged to give an n = 1.
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