Briefly, 100 µL of cells at 1 million cells per milliliter was plated on 96-well plates. Cells in each well were transfected with a plasmid mixture of (1) 200 ng of pG5luc, which contains the firefly luciferase gene whose expression is controlled by UAS sequences; (2) 2 ng of pRL-hsp70, which contains a constitutively expressed Renilla luciferase gene; and (3) 50 ng of pAc5.1 vector containing either Gal4DBD-tagged NSL3 protein or Gal4DBD alone. Transfections were performed with X-tremeGENE DNA transfection reagents (Roche). After 2 d of incubation, cells were lysed (dual-luciferase kit, Promega), and luminescence was measured by using a Mithras plate reader (Berthold). For further details, see the Supplemental Material .
Mithras plate reader
Manufactured by Berthold Technologies
Sourced in United States, Germany
The Mithras plate reader is a versatile and reliable instrument used for high-throughput analysis in life science research. It is designed to measure absorbance, fluorescence, and luminescence in multi-well microplates. The Mithras plate reader provides accurate and reproducible results, making it a valuable tool for a wide range of applications, including cell-based assays, enzyme activity measurements, and drug discovery screening.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase kit, by Promega (1 mentions)
X tremegene dna transfection reagent, by Roche (1 mentions)
Polarstar optima plate reader, by BMG Labtech (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Dual glo reagent, by Promega (1 mentions)
Celltiter glo, by Promega (1 mentions)
Atplite luminescence assay system, by PerkinElmer (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Bright glo reagent, by Promega (1 mentions)
Prism 5, by GraphPad (1 mentions)
Jetpei reagent, by Polyplus Transfection (1 mentions)
9 protocols using mithras plate reader
1
Gal4DBD-NSL3 protein reporter assay
2
BRET Saturation Experiments in HEK-293 Cells
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For BRET saturation experiments, HEK‐293 cells transiently transfected with a constant amount of cDNA encoding the Rluc constructs and increasing amounts of YFP tagged proteins were rapidly washed twice in PBS, detached and resuspended in Hank's balanced salt solution (HBSS) buffer (137 mM 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, pH 7.4), containing 10 mM glucose and processed for BRET determinations using a POLARstar Optima plate‐reader (BMG Labtech, Durham, NC, USA; Ciruela et al, 2015) or Mithras plate reader (Berthold Technologies; Cecon et al, 2015 ).
3
Redirected Lysis Assay for BiTE/Triplebody
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To quantitate cell-mediated cytolysis (referred to as redirected lysis, RDL) induced by the BiTE or triplebody proteins, target cells were pre-labelled with Calcein AM (Life Technologies) and mixed with effector cells in RPMI 1640 GlutaMAX medium supplemented with 10% fetal calf serum at an E : T ratio of 10 : 1 unless otherwise stated. Either MNCs expanded ex vivo for 20 days and pre-stimulated with anti-CD3 mAb OKT3 and IL-2,[11 (link), 56 (link)] or untouched T cells isolated via magnetic separation, were used as effector cells. After addition of different antibody-derived proteins to 200 μL reaction volume in round-bottom 96-well plates, the reactions were incubated at 37 °C with 5% CO2. Calcein release was determined by measuring the fluorescence intensity (relative light units, RLU) in the supernatant with a Berthold Mithras plate reader (Berthold technologies, Bad Wildbad, Germany). Maximum lysis was achieved by addition of 50 μL of a solution containing 10% Triton X-100 in RPMI 1640 GlutaMAX medium supplemented with 10% fetal calf serum and 1% Penicillin/Streptomycin. Specific lysis was calculated as follows:
% specific Lysis = 100*[RLU (sample) − RLU (background)] / [RLU (maximal lysis) − RLU (background)]
4
Quantification of IFN-β in VSV-infected HFFs
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IFN-β production of VSV-infected HFFs was detected using the LumiKine human IFN-β ELISA kit (#lumi-hifnbv2; Invivogen) according to the manufacturer’s instructions. Lucia luciferase signals were assessed in a Mithras plate reader (Berthold Technologies GmbH & Co. KG).
5
Dual-Glo Luciferase Assay Protocol
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Dual-Glo Reagents were purchased from Promega and used per the manufacturer's instruction. Assays were measured using a Mithras plate reader from Berthold and expressed as a ratio of Firefly/Renilla luciferase RLU signal.
6
Antibody-Drug Conjugates Targeting IGF-1R
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Example 23
The five IGF-1R antibodies were shown to be rapidly internalized into lysosomes and to have a lower binding capacity into acidic environments. In that respect, those Abs had all properties to be used as ADCs. Thus, the five chimeric anti-IGF-1R antibodies were coupled with three different compounds (G-13; E-13 and F-63). The drug antibody ratio of those ADCs was about 4. In order to evaluate the non specific cytotoxicity, an irrelevant chimeric antibody c9G4 was also coupled with those compounds at the same DAR. MCF-7 cells were incubated with increasing concentrations of each ADCs at 37° C. for 6 days in complete culture medium. Cell viability was assessed using a luminescent cell viability assay (CELLTITER-GLO®, PROMEGA®). Luminescent signal was read using a the Mithras plate reader (Berthold Technologies). The irrelevant chimeric antibody c9G4 coupled with either E-13, G-13 or F-63 showed no or modest cytotoxic activity on MCF-7 cells (
7
Intracellular ATP and Amino Acid Assays
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Cells were seeded in 6‐well plates and treated with the respective dsRNAs as described above. After 3 days, the cells were resuspended, counted, and seeded in a 24‐well plate at 750,000 cells/well. After an overnight incubation at 25°C, the cells were starved for amino acids for 30 min and then lysed in the plate with 80 μl of ATP lysis buffer (ATPlite™ Luminescence Assay System, Perkin Elmer #6016941); 3 μl of the lysate was mixed with 100 μl of the substrate, and after 10‐min incubation at room temperature in the dark, the signal was read in a Mithras plate reader (Berthold Technologies). The signal was normalized to total protein levels measured by Bradford assay (BIORAD).
Intracellular amino acid measurements were performed as described in Demetriades et al (2014 ). Five biological replicates per condition were analyzed.
Intracellular amino acid measurements were performed as described in Demetriades et al (
8
Cell Viability Assay Protocol
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RPE-1 were seeded at 4000 cell/well in 96-well plate with 200 μl of complete medium. After 24 h, medium was replaced by fresh medium complemented with different drugs at varying doses. Cell viability, after indicated times of drug exposure, was quantified using CellTiter AQ one solution MTS reagent (G5421, Promega, Madison, WI) according to manufacturer's instructions and measured using a Mithras plate reader (Berthold).
9
In Vitro TGR5 Activation Assay
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In vitro TGR5 Assay. TGR5 activation by compounds and subsequent increase in intracellular cAMP were evaluated using a luciferase reporter gene assay. Human embryonic kidney (HEK) 293 cells were transiently co-transfected with pCMV tag4b-TGR5h (to determine hTGR5 activation) or pCMV AC6-TGR5m (to determine mTGR5 activation) expression plasmids and the pCRE TA-Luciferase reporter plasmid using the JET PEI reagent (Polyplus transfection).
Transfected cells were seeded in 96-well plates and incubated overnight with the compounds at increasing concentrations in duplicate. Lithocholic acid (LCA) at 10 µM was used as a positive reference compound. The cAMP-dependent luciferase expression was followed using the BrightGlo reagent according to the manufacturer (Promega) instructions. Luminescence was measured with a Mithras plate reader (Berthold). Data were expressed as percentage of the 10µM LCA value and EC 50 values were calculated using XL fit 5 software or GraphPad Prism 5.
Concentration-response curves were fitted by a nonlinear regression analysis to a 4 parameters logistic equation.
Transfected cells were seeded in 96-well plates and incubated overnight with the compounds at increasing concentrations in duplicate. Lithocholic acid (LCA) at 10 µM was used as a positive reference compound. The cAMP-dependent luciferase expression was followed using the BrightGlo reagent according to the manufacturer (Promega) instructions. Luminescence was measured with a Mithras plate reader (Berthold). Data were expressed as percentage of the 10µM LCA value and EC 50 values were calculated using XL fit 5 software or GraphPad Prism 5.
Concentration-response curves were fitted by a nonlinear regression analysis to a 4 parameters logistic equation.
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