Viral RNA was isolated using RNeasy® Plus Universal Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. Quantification of viral RNA was performed using a real-time quantitative polymerase chain reaction (RT-qPCR) assay specific for the HTNV nucleocapsid coding region in a LigthCycler® 96 (Roche, Mannheim, Germany) following the manufacturer’s instructions. Cells treated in parallel with the drugs were analyzed for viability using the CellTiter-Glo® Assay System (Promega).
Celltiter glo assay system
Manufactured by Promega
Sourced in United States
The CellTiter-Glo assay system is a cell-based luminescent assay that quantifies the amount of ATP present in metabolically active cells. The assay provides a quantitative measure of cell viability and cytotoxicity.
Automatically generated - may contain errors
Lab products found in correlation
Realtime glo mt viability assay system, by Promega (2 mentions)
Glomax multi jr single tube multimode reader, by Promega (2 mentions)
Flexitube sirna, by Qiagen (2 mentions)
Rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Ligthcycler 96, by Roche (1 mentions)
Rneasy plus universal mini kit, by Qiagen (1 mentions)
Monensin sodium salt, by Merck Group (1 mentions)
Tetrandrine, by Merck Group (1 mentions)
5 n ethyl n isopropyl amiloride eipa, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Flextube sirna, by Qiagen (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
M1000, by Tecan (1 mentions)
Bafa1, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Hepatocyte growth factor (hgf), by Merck Group (1 mentions)
Ln405, by American Type Culture Collection (1 mentions)
Tristar lb 941, by Berthold Technologies (1 mentions)
Cytotox one reagent, by Promega (1 mentions)
18 protocols using celltiter glo assay system
1
HTNV RNA Quantification and Cell Viability
2
High-throughput Screening of Antiviral Compounds
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pWRG/HTNV-M [12 (link)] was kindly provided by Connie. S. Schmaljohn (U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA). The expression plasmid pI18 for the GPC of ANDV strain CHI-7913 was kindly provided by Nicole Tischler (Molecular Virology Laboratory, Fundación Ciencia & Vida, Santiago, Chile) and has been described previously [13 (link)]. Expression plasmids encoding Lassa virus GPC strain Josiah and VSV-G were reported previously [8 (link)]. The expression vector encoding Ebola virus strain Makona was kindly provided by Mark Page (The National Institute for Biological Standards and Control, South Mimms, UK).
The phytochemical library was from Prestwick Chemicals (stock concentration 10 mM in DMSO, Illkrich Graffenstaden, France). The selected positive hits monensin sodium salt, rotenone, tetrandrine, as well as 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were from Sigma-Aldrich (Buchs, Switzerland), nitrarine dihydrochloride was provided by Latoxan (Portes lès Valence, France), and emetine dihydrochloride was from EMD Millipore (Darmstadt, Germany). The CellTiter-Glo® Assay System was obtained from Promega (Madison, WI, USA).
The phytochemical library was from Prestwick Chemicals (stock concentration 10 mM in DMSO, Illkrich Graffenstaden, France). The selected positive hits monensin sodium salt, rotenone, tetrandrine, as well as 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were from Sigma-Aldrich (Buchs, Switzerland), nitrarine dihydrochloride was provided by Latoxan (Portes lès Valence, France), and emetine dihydrochloride was from EMD Millipore (Darmstadt, Germany). The CellTiter-Glo® Assay System was obtained from Promega (Madison, WI, USA).
3
Cytotoxicity Assessment of Co(OH)2 Nanostructures
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Co(OH)2 NS were tested on
different cell lines that were seeded in 96-multiwell plates at a
density of 1 × 103 (cancer cells) or 8 × 103 (MRC-5 cells). Cell viability was measured using the CellTiter-Glo
assay system according to the manufacturer’s instructions4 (link) (Promega, Madison, Wisconsin) after 96 h. Six
serial dilutions 1:10 of the compounds were utilized to calculate
the IC50 values with GraphPad software utilizing the nonlinear
regression method.
Organoids were plated as single cells as
possible (around 1000 cells) in five replicates and treated with six
serial dilutions 1:10 of the compounds and analyzed after 96 h as
cell lines.
different cell lines that were seeded in 96-multiwell plates at a
density of 1 × 103 (cancer cells) or 8 × 103 (MRC-5 cells). Cell viability was measured using the CellTiter-Glo
assay system according to the manufacturer’s instructions4 (link) (Promega, Madison, Wisconsin) after 96 h. Six
serial dilutions 1:10 of the compounds were utilized to calculate
the IC50 values with GraphPad software utilizing the nonlinear
regression method.
Organoids were plated as single cells as
possible (around 1000 cells) in five replicates and treated with six
serial dilutions 1:10 of the compounds and analyzed after 96 h as
cell lines.
4
Screening for Anti-Influenza Host Factors
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siRNA treatment procedure, cell viability, and virus titer determination are described in detail in Watanabe et al. (32 (link)). Briefly, two siRNAs per candidate gene were selected from a predesigned genome-wide human siRNA library (FlexTube siRNA; Qiagen). AllStars Negative Control siRNA (Qiagen) was used as a negative control. The siRNA against the NP gene of WSN virus (GGA UCU UAU UUC UUC GGA GUU) purchased from Sigma-Aldrich was used as a positive control. HEK293 cells were transfected twice with 25 nM (final concentration, 50 nM) siRNA duplexes using RNAiMAX (Invitrogen). At 24 h after the second transfection, cell viability was determined using the CellTiter-Glo assay system (Promega) following the manufacturer’s instructions. To assess influenza virus replication, two parallel sets of siRNA-transfected cells were infected with 50 plaque-forming units (PFU) of WSN virus per well of a 24-well tissue culture plate at 24 h after the second siRNA transfection. At 48 h postinfection, supernatants were harvested, and virus titers were determined by plaque assay in MDCK cells.
5
Cell Viability Assay with CKD-581 and SAHA
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Cells cultured in 96-well plates were treated with vehicle (0.1% DMSO), CKD-581 or SAHA. Cell viability was determined by CellTiter-Glo assay system (Promega) 72 h after compound treatment. Luminescence was determined using FlexStation 3 (Molecular Devices, San Jose, CA, USA).
6
Evaluating AgNP Cytotoxicity on Cell Lines
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To investigate the AgNP on cell viability, A2780, A2780cis, SK-OV-3, MDA-MB-231, U-87MG, MCF-7 were seeded in 96 multiwell plates at the concentration of 1x103 and MRC-5, W1-38 8x103 per well and treated with six concentrations (0.001, 0.01, 0.1, 1, 10 and 100 μg) of AgNP and its precursors AgNO3 and SNP. After 96h cell viability was quantified using the CellTiter-Glo® assay system according to the manufacturer’s instructions (Promega, Madison, WI, USA). Luminescence was recorded in a Tecan M1000 instrument (Tecan, Mannedorf, Switzerland). Experiments performed in triplicates and IC50 values were determined from non-linear regression method using GraphPad prism software.
7
Glycosylated Dystroglycan Immunoassay Protocol
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Monoclonal antibodies to alpha-dystroglycan (IIH6) for glycosylated DG have been described previously [69 (link)]. Purified polyclonal goat IgG anti human Axl (#AF154) and anti-human HGFR (for IFA, #AF276) were purchased from R&D Systems (Minneapolis, MN, USA). Monoclonal 113 anti-LCMV NP antibody has been described previously [70 (link)]. For Western blot, anti-human HGFR and anti-human phospho-HGFR (10/2017 and 08/2017, respectively) were purchased from Cell Signalling (Leiden, The Netherlands). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Dako, and phycoerythrin (PE)-conjugated secondary antibodies from Biolegend. The CellTiter-Glo assay system was purchased from Promega (Madison, WI, USA). Dextran, Alexa FluorTM 488 (10 kDa), and NucBlueTM Fixed cells ReadyProbesTM Reagent were purchased from Thermo Fisher (Waltham, MA, USA). R428 was obtained from Selleckem (Houston, TX, USA). EMD, EIPA, Rib, BafA1, and HGF were purchased from Sigma (St. Louis, MO, USA).
8
Quantify Cell Viability via Luminescence
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Assessment of cell viability was made by measuring the cell titre at the time points detailed. Cell titre was determined using both manual haemocytometer counts (without trypan blue) and the CellTiter-Glo assay system (#G7570, Promega) following the manufacturer’s protocol and luciferase readings measured using a GloMax-Multi Jr Single-Tube Multimode Reader (E6076, Promega) using the Cell-titre-Glo default settings. Viability was determined using RealTime Glo MT Viability assay system (#G9711, Promega), following all steps of the manufacturer’s protocol and readings taken using a GloMax-Multi Jr Single-Tube Multimode Reader (E6076, Promega) using the Cell-titre-Glo default settings.
9
Cytotoxicity Screening of P-Analog Compounds
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HEK293 and human glioblastoma cell lines LN405, T98G, U87 were obtained from the American Type Culture collection (LGC Standards GmbH; Wesel, Germany). Cells at a density of 2.5 × 104 cells/ml, 200 μl per well, were seeded on microplates (Greiner, μClear, black clear bottom) in DMEM (4.5 g/L glucose, 10% FCS, 2 mM Glutamax, containing antibiotics). Medium was exchanged 24 h later for 100 μl per well of Hanks’ solution (HBSS) (1 g/L glucose, 0.37 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 4.2 mM NaHCO3). P-analogs of pyruvate were added at different concentrations (0.2, 0.5, 1.0, 10 and 20 mM). 5 h later ATP levels were determined using the CellTiterGlo assay system (Promega, Heidelberg, Germany) according to manufacturer's recommendations as described previously [40 (link)]. Concentration dependence data were obtained by averaging luminescence from six wells, and % of the ATP levels in the treated vs control cells were used to characterize the effects of the analogs on cellular viability.
10
Antiviral Activity and Cytotoxicity Assay
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Vero E6 cells were pre-treated with increasing concentrations of EIPA, monensin sodium salt, emetine dihydrochloride and tetrandrine for 30–45 min at 37 °C, followed by HTNV (strain 76/118) infection for 1 h at 37 °C in presence of the drug. Cells were subsequently washed once with MEM 2% FCS and incubated with MEM 2% FCS containing appropriate drug concentrations for 2 days.
To calculate the therapeutic index (TI), confluent monolayers of Vero E6 cells were treated with EIPA, tetrandrine, and emetine dihydrochloride starting at 100 µM concentration, followed by a ~1.5-fold dilution for two days. After 48 h, cells were treated with 1% triton-100 (diluted in water) for 15 min at 37 °C in order to totally lyse the cells and obtain the background values. Cell viability was then measured using CellTiter-Glo® Assay System (Promega). The median toxic dose (TD50) was calculated based on the cell viability values, while the median effective dose (ED50) on cell infectivity with HTNV. Therapeutic index was finally calculated as follows: TD50/ED50.
To calculate the therapeutic index (TI), confluent monolayers of Vero E6 cells were treated with EIPA, tetrandrine, and emetine dihydrochloride starting at 100 µM concentration, followed by a ~1.5-fold dilution for two days. After 48 h, cells were treated with 1% triton-100 (diluted in water) for 15 min at 37 °C in order to totally lyse the cells and obtain the background values. Cell viability was then measured using CellTiter-Glo® Assay System (Promega). The median toxic dose (TD50) was calculated based on the cell viability values, while the median effective dose (ED50) on cell infectivity with HTNV. Therapeutic index was finally calculated as follows: TD50/ED50.
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