Levels of glutathione (GSH) and oxidized glutathione (GSSG) were assess using a GSH-Glo™ Glutathione Assay (Promega, Madison, WI). The protocol (384-well format) was modified from the manufacturer’s protocol (96-well format, Promega V6911). In brief, 2.5 µl of unclarified lung lavage samples (in duplicate) were treated with 2.5 µl GSH-Glo reagent with or without TCEP (final concentration = 1 mM). Following a 30 min incubation at room temperature, luciferin detection reagent (5 µl) was added, and luminescence signals were recorded on a plate reader (PerkinElmer EnVision 2104, 700 nm, 0.5 s exposure) 15 min later. For reactive oxygen species (ROS), samples were tested using a ROS-Glo Assay (Promega). Lung lavage samples were prepared as described above for GSH-Glo assays. Following a 60 min incubation at room temperature, luciferin detection reagent (5 µl) was added, and luminescence signals were recorded on a plate reader (PerkinElmer EnVision 2104, 700 nm, 0.5 s exposure) 20 min later.
Envision 2104
Manufactured by PerkinElmer
Sourced in United States
The Envision 2104 is a multimode plate reader designed for high-throughput screening and cell-based assays. It offers a range of detection modes, including absorbance, fluorescence, luminescence, and time-resolved fluorescence. The Envision 2104 is capable of reading 6- to 384-well microplates and can be configured with up to four detection modules to accommodate various assay requirements.
Automatically generated - may contain errors
Lab products found in correlation
Janus workstation, by PerkinElmer (9 mentions)
Alphaplate 384, by PerkinElmer (9 mentions)
Celltiter glo, by Promega (7 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (2 mentions)
Bright glo luciferase assay system, by Promega (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Quanti luc coelenterazine based luminescence assay detection reagent, by InvivoGen (2 mentions)
Celltiter glo assay kit, by Promega (2 mentions)
Glucose assay kit, by Abcam (2 mentions)
Optiplate, by PerkinElmer (2 mentions)
Dual glo luciferase system, by Promega (2 mentions)
Prism 5, by GraphPad (2 mentions)
Free fatty acid uptake assay kit, by Abcam (2 mentions)
Prism 8, by GraphPad (2 mentions)
Celltiter glo assay, by Promega (2 mentions)
Gst dnmt3a, by Active Motif (2 mentions)
Al109m, by PerkinElmer (2 mentions)
Alamarblue reagent, by Thermo Fisher Scientific (2 mentions)
Ros glo assay, by Promega (1 mentions)
Gsh glo glutathione assay, by Promega (1 mentions)
100 protocols using envision 2104
1
Glutathione and ROS Assessment in Lung Lavage
2
Caspase 3/7 Fluorescence Apoptosis Assay
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Apoptosis was measured with a caspase 3/7 fluorescence assay (Promega, Madison WI). For MCF10F 100 μL caspase 3/7 reagent was added per well and for HCC70 and SKBR3 40 μL of caspase 3/7 reagent was added per well. The plates were then incubated for 30 min at 37 °C and read by luciferase intensity using the Wallac Envision 2104 Multi-label Detector/plate reader with a 96-well aperture (Perkin Elmer, Waltham MA).
3
AlphaScreen Assay for Brd4 Inhibitor
4
AlphaScreen Assay for BRD9 Ligand Screening
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Example 14
Assays are performed with minimal modifications from the manufacturer's protocol (PerkinElmer, USA). All reagents are diluted in 50 mM HEPES, 150 mMNaCl, 0.1% w/v BSA, 0.01% w/v Tween20, pH 7.5 and allowed to equilibrate to room temperature prior to addition to plates. After addition of Alpha beads to master solutions all subsequent steps are performed under low light conditions. A 2× solution of components with final concentrations of BRD9 at 40 nM, Ni-coated Acceptor Bead at 10 μg/mL, and 20 nM biotinylated-BRD9 targeting ligand is added in 10 μL to 384-well plates (AlphaPlate-384, PerkinElmer, USA). Plates are spun down at 150×g, 100 nL of compound in DMSO from stock plates are added by pin transfer using a Janus Workstation (PerkinElmer, USA). The streptavidin-coated donor beads (10 μg/mL final) are added as with previous the solution in a 2×, 10 μL volume. Following this addition, plates are sealed with foil to prevent light exposure and evaporation. The plates are spun down again at 150×g. Plates are incubated at room temperature for 1 hour and then read on an Envision 2104 (PerkinElmer, USA) using the manufacturer's protocol. The data are analyzed using PRISM Graphpad v6 to obtain IC50 values.
5
ATP Luminometric Quantification
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ATP levels were assayed luminometrically using the ATPLITE 1 STEP (Perkin Elmer, Waltham, MA, USA, 6016731) according to the procedure recommended by the manufacturer’s instructions. 4 × 104 cells were seeded for 24 h. The ATP content was measured considering the total (10 mM glucose), the glycolytic (10 mM glucose plus 2.5 mg/mL oligomycin; G + O) and the oxidative ATP production (50 mM 2-deoxy-d -glucose plus 5 mM pyruvate; 2DG + P). Glucose (Sigma Aldrich, G8270), Oligomycin A (Sigma Aldrich, 75351), Sodium Pyruvate (Sigma Aldrich, P2256). Briefly, cells incubated for 2 h in record solution accordingly to report elsewhere [43 (link)]. Sample’s luminescence was measured using a Perkin Elmer Envision 2104 multilabel reader.
6
Transient HeLa Cell Transfection and Infection
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HeLa cells were transfected with 75 ng of expression plasmid and 425 ng of puromycin resistance plasmid. At 24 h posttransfection, cells were infected with the NL-Luc virus stock (1:27 dilution) in Opti-MEM reduced serum medium (Life Technologies) plus 8 μg/ml Polybrene (Sigma-Aldrich) and 1 μg/ml puromycin (InvivoGen) for 90 min at 37°C and 5% CO2. The inoculum was removed, and the cells were washed with PBS and incubated at 37°C and 5% CO2 for 48 h in growth medium (complete DMEM) with 20% FCS plus 1 μg/ml puromycin to ensure survival and expression of reporters in transfected cells only. Luciferase readout was performed with the BrightGlo luciferase assay system (Promega) on an Envision 2104 plate reader (Perkin Elmer) according to the manufacturer’s instructions.
7
CRISPR Exon Skipping Reporter Assay
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In a 96-well plate format, 100 ng of pPV-EF1α-Cas7-Cas5-Cas8-iPA, pPV-EF1α-Cas11-Cas6-Cas3-iPA and crRNA expression plasmids were co-transfected with 100 ng of the luciferase-based exon skipping reporter plasmid pPV-EF1α-Luc2(V323I)-hDMD-Ex45[+](4 kb)-iPA and 20 ng of Renilla luciferase plasmid phRL-TK as a transfection control. For the SpCas9 control, 200 ng of pPV-EF1α-Cas9-iPA and 100 ng of sgRNA (or 50 ng each for two sgRNAs) expression plasmids were used. These plasmids were diluted in 25 µl of Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific), mixed with 1 µl of Lipofectamine 2000, diluted in 25 µl of Opti-MEM Reduced Serum Medium and incubated for 30 min at room temperature in each well of a 96-well plate. 293T cells were dissociated by trypsin-EDTA and seeded into each well at 6 × 104 cells per 100 µl per well. After 48 h cultivation, luciferase activity was measured using a Dual-Glo Luciferase Assay Kit (Promega) and Envision 2104 (Perkin Elmer) plate reader following the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase activity.
8
Cytotoxicity Evaluation of Compounds on Primary Human Hepatocytes
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Potential cytotoxicity of all compounds in each cell system was determined in pilot experiments in order to identify the highest compound concentration that did not induce significant toxicity, as judged by cell growth and morphological observation under light microscopy. The cytotoxicity of compounds on cryopreserved PHHs was further determined by using a CellTiter-Glo luminescent cell viability assay, which is based on measurement of cellular ATP content (Promega, Madison, WI). Briefly, PHHs (BioIVT) were seeded into collagen-coated 96-well plates at a density of 3 × 104 cells per well and then incubated overnight under standard conditions to allow cell attachment. The culture medium was then removed, and the cells were exposed to the indicated concentrations of the compounds for 72 h. Cell lysate was then prepared according to the manufacturer’s instructions, and luminescence was read on a microplate reader (Envision 2104; Perkin-Elmer). Luminescence data were converted to growth fraction by comparison to the luminescence readout for the untreated control, and 50% inhibitory concentrations were determined from the graphical data (Fig. S4 ).
9
High-Throughput Viability Assay for Cells
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The cells were plated at 10,000 cells per well in a clear bottom black 96-well plate (Greiner Cat. No. 655090) and a white 96-well plate (Greiner Cat. No. 655083) then they were placed in an incubator. After 24 hr, the plates were removed from the incubator and treated with drugs using the HP D300 Digital Dispenser. After the targeted drug treatment times, 100 µL of Cell-Titer-Glo (Promega Corp.) was added to the wells in the white 96-well plate and shaken at 500 rpm for 5 min. The plate was then read by the Perkin Elmer Envision 2104 using an enhanced luminescence protocol to count the number of raw luminescent units per well. For the black clear bottom 96-well plates, the plate was spun at 300 g for 5 min and all the cell media was removed. Methanol was then added at 200 µL per well and let sit at room temperature for 15 min. The methanol was removed from the wells and 200 µL of PBS with Hoechst 33342 nucleic acid stain at a final concentration of 1 µG/mL was then added to the wells. The plates were then imaged with the GE Healthcare IN Cell Analyzer 2000 that is equipped with a CCD camera. The IN Cell Analyzer software was used to count the number of cells detected in each well to calculate the viability. Three replicates were used for the combination experiments and two replicates for the dose experiments.
10
Cell Viability Assay for W12 and C33A Cells
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