Antibody titers against Anc80L65 in CSF and serum were determined through neutralization assays3 (link). Using a 96-well format, heat-inactivated CSF or serum samples (collected as described above) were serially diluted in serum-free medium (Life Technologies, Carlsbad, CA), and then treated with Anc80L65-luciferase (106 GC/well) for 1 hour at 37 °C. The sample/Anc80L65-luciferase mix was then transferred onto HEK293 cells, which were treated with adenovirus (MOI 20) the day before. After 1 hour at 37 °C, diluted serum medium (1 part serum-free, 2 parts with serum) was added to each well. Two days later, the cells were treated with lysis buffer (Promega, Madison, WI) and frozen at −80 °C for 30 minutes. The cells were then thawed at 37 °C for 15 minutes before being treated with substrate buffer (Tris-HCl, MgCl2, ATP [Life Technologies, Carlsbad, CA], D-Luciferin [Caliper Life Sciences, Hopkinton, MA]). Luminescence output was read using the Synergy BioTek Plate Reader (BioTek, Winooski, VT).
Biotek synergy plate reader
Manufactured by Agilent Technologies
Sourced in United States
The BioTek Synergy plate reader is a multi-mode microplate reader designed for a variety of detection technologies, including absorbance, fluorescence, and luminescence. It is capable of reading 96- and 384-well microplates.
Automatically generated - may contain errors
Lab products found in correlation
Lysis buffer, by Promega (3 mentions)
Serum free medium, by Thermo Fisher Scientific (3 mentions)
Tris hcl, by Thermo Fisher Scientific (2 mentions)
Mgcl2, by Thermo Fisher Scientific (2 mentions)
Lanthascreen tr fret car coactivator binding assay, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Menadione, by Merck Group (1 mentions)
Pci neo vector, by Promega (1 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Nefa kit, by Randox (1 mentions)
Bovine igg1 elisa quantitation set, by Fortis Life Sciences (1 mentions)
Neuronal extraction buffer, by Thermo Fisher Scientific (1 mentions)
Bca assay kit, by Abcam (1 mentions)
Dual luciferase reporter plasmid, by Promega (1 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
96 well plate, by Corning (1 mentions)
Cell lysis buffer, by Promega (1 mentions)
Growth factor reduced matrigel matrix, by Corning (1 mentions)
14 protocols using biotek synergy plate reader
1
Anc80L65 Neutralization Assay in CSF and Serum
2
LanthaScreen TR-FRET Receptor Binding Assay
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The LanthaScreen® TR-FRET CAR Coactivator Binding Assay (Thermo Fisher Scientific, Waltham, MA, United States) was performed according to the manufacturer’s protocol with some modifications described in our previous paper (Carazo Fernandez et al., 2015 (link)). The LanthaScreen® TR-FRET GR assay was performed following the protocol of the manufacturer. Results were obtained after fluorescence signal analysis with appropriate filters using the Synergy Biotek plate reader (BioTek Instruments Inc., Winooski, VT, United States). Results were calculated as relative fold interaction of a nuclear receptor with the coactivator fragment to the control (vehicle-treated, DMSO 0.1%) signal set to 1. Data are presented as the means and SD of three different experiments (n = 3) performed in quadruplicates.
3
Anc80L65 Neutralization Assay in CSF and Serum
4
CAR Coactivator Binding Assay
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The LanthaScreen® TR-FRET CAR Coactivator Binding Assay (Thermo Fisher Scientific, Waltham, MA, USA) was used with some modifications to the manufacturers’ protocol reported in our previous paper [27 (link)]. In order to allow better interaction of benzodiazepines with the CAR LBD, incubation time was extended to 3 h. For fluorescence measurement, the Synergy Biotek plate reader was used with appropriate filters (BioTek Instruments Inc., Winooski, VT, USA). Results were calculated as an interaction of the CAR-LBD with the coactivator fragment relative to the control (DMSO 0.1%). Data are presented as the means and SD of a technical quadruplicate (n = 4).
5
Overexpression of XIAP Protects 661W Cells
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The transformed cone photoreceptor cell line, 661W, was kindly provided by Dr. M. Al-Ubaiddi.38 (link) The 661W cells were transfected with the full-length coding sequence of human XIAP, which was generated from pCM-SPORT6-XIAP (Origene, Rockville, MD) digested with SalI and NotI to isolate the XIAP coding sequence, ligated into the pCI-neo vector (Promega, Madison, WI). Transfections were conducted using Lipofectamine 2000 (ThermoFisher Scientific), according to the manufacturer's instructions. Control cell lines were transfected in a similar manner with the pCI-neo empty vector. Stably expressing cells were grown under G418 selection. All cells were used below passage 15, and XIAP expression was monitored in each assay by Western blot or quantitative RT-PCR.
For the death assays, cells were plated onto 96-well plates at 60% confluency and incubated with 1 mM hydrogen peroxide (Sigma Aldrich) for 32 hours or 15 µM menadione (Sigma Aldrich) for 4 hours. Cell viability was measured using the AlamarBlue Cell Viability Reagent (Invitrogen) and a Synergy BioTek plate reader (BioTek Instruments, Inc.) as per manufacturer's directions. Read-out measurements of treated cells were divided by vehicle control-treated cells to determine final viability. Three individual plates with five replicates each were averaged.
For the death assays, cells were plated onto 96-well plates at 60% confluency and incubated with 1 mM hydrogen peroxide (Sigma Aldrich) for 32 hours or 15 µM menadione (Sigma Aldrich) for 4 hours. Cell viability was measured using the AlamarBlue Cell Viability Reagent (Invitrogen) and a Synergy BioTek plate reader (BioTek Instruments, Inc.) as per manufacturer's directions. Read-out measurements of treated cells were divided by vehicle control-treated cells to determine final viability. Three individual plates with five replicates each were averaged.
6
Neutralization Assay for Anc80L65 Antibody
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Antibody titers against Anc80L65 in CSF and serum were determined through neutralization assays. Using a 96-well format, heat-inactivated CSF or serum samples (collected as described above) were serially diluted in serum-free medium (Life Technologies), and then treated with Anc80L65-luciferase (107 GC/well) for 1 h at 37 °C. The sample/Anc80L65-luciferase mix was then transferred onto HEK293 cells (10,000 cells/well), which had been treated with adenovirus (MOI 100) the day before. After 1 h at 37 °C, diluted serum medium (1 part serum-free, 2 parts serum) was added to each well. Two days later, the cells were treated with lysis buffer (Promega) and frozen at −80 °C for 30 min. The cells were then thawed at 37 °C for 15 min before being treated with substrate buffer (Tris-HCl, MgCl2, ATP) (Life Technologies) and D-luciferin (Caliper Life Sciences). Luminescence output was read using the Synergy BioTek Plate Reader (BioTek). Data Analysis was performed using Microsoft Excel.
7
Plasma Analyte Concentration Quantification
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The plasma glucose and nonesterified fatty acids (NEFA) concentrations were determined using glucose kit and NEFA kit (Randox Laboratories Ltd, UK), respectively. An automated RX series Clinical Chemistry Analysers (Randox Laboratories Ltd, UK) was used for all measurements. The plasma IgG1 concentrations were detected using a bovine IgG1 ELISA Quantitation set (Bethyl Laboratories, USA) according to the manufacturer’s protocol. Plasma was diluted in Tris-buffered saline (TBS)-Tween to a final dilution factor of 4 × 104. All dilutions were duplicated. Absorbance was read using a BioTek Synergy plate reader (BioTek Instruments, Inc., USA) at a wavelength of 450 nm.
8
Cell Viability Assay with AlamarBlue
9
Cell Viability Assay with C830 and C800
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Cell viability was determined using the MTT method. Briefly, SK-MEL, OU-31, PC-3, OVCAR, MCF-7, HT-29 and HOP-92 cells were seeded in 96-well plates at a density of 8000 cells per well and treated with 0.5 μM C830 and C800 for 24 h. MTT was added to culture medium four hours before incubations ended. Finally, reduced MTT was dissolved in DMSO and absorbance was determined at 570 and 670 nm (reference) using a Bio-Tek Synergy plate reader (Bio-Tek). For each cell line three experiments with n = 8 were performed.
10
Neuronal Protein Extraction and Analysis
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Neuronal extraction buffer (87792, Thermo Fisher Scientific) mixed with 1X protease inhibitor cocktail and 0.1 mM PMSF was used to solubilize cultured cortical cells and brain tissues. For protein quantification, BCA assay kit (BioVision), BioTek Synergy plate reader and Gen5 software (BioTek Instruments, Winooski, VT) were used. Immunoblotting was conducted using a 12 % SDS gradient polyacrylamide gel followed by transfer on PVDF membrane. The primary antibodies used in this paper are as follows: ATPase5β (1:1000, Sigma-Aldrich HPA001520), β-actin (1:5000, BioRad HCA147P), α-tubulin (1:2000, Sigma-Aldrich T6074), Sirt3 (1:1000, Cell Signaling Technology 5490S), and anti-acetyl lysine (1:1000, Cell Signaling Technology 9441S).
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