Envision microplate reader

Manufactured by PerkinElmer

Sourced in United States

The EnVision microplate reader is a versatile instrument designed for a wide range of microplate-based applications. It utilizes advanced optical components and detection technologies to provide accurate and reliable measurements of fluorescence, luminescence, and absorbance in multi-well microplates.

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Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions) Alamar blue reagent, by HiMedia (3 mentions) Dual glo luciferase assay system, by Promega (3 mentions) Prism 7, by GraphPad (3 mentions) Pen strep glutamine, by Thermo Fisher Scientific (2 mentions) Bright glo, by Promega (2 mentions) Modified griess reagent, by Merck Group (2 mentions) Elisa kit, by BD (2 mentions) Goat anti mouse igg hrp, by Agilent Technologies (2 mentions) Tmb substrate, by Merck Group (2 mentions) Human γ377 395 peptide, by Thermo Fisher Scientific (2 mentions) Maxisorp elisa plate, by Thermo Fisher Scientific (2 mentions) D luciferin salt, by PerkinElmer (2 mentions) Collagen 1 coated 384 well plate, by Greiner (2 mentions) Human c5a elisa kit 2, by BD (2 mentions) Human c3a elisa kit, by BD (2 mentions) Prism v8, by GraphPad (2 mentions) Celltiter glo luminescent cell viability assay, by Promega (2 mentions) Lance ultra camp kit, by PerkinElmer (2 mentions) Cck 8 kit, by Dojindo Laboratories (2 mentions)

148 protocols using envision microplate reader

Colorimetric Assay for MTH1 Activity

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On the basis of the ability of MTH1 in converting dGTP to dGMP and pyrophosphate, and the latter can then be hydrolyzed to inorganic pyrophosphate by inorganic pyrophosphatase, the MTH1 activity was designed to measure the product of inorganic pyrophosphate, after its reaction with malachite green and ammonium molybdate(Fig. 2l), the green associated complex was measured at 630 nm by PerkinElmer Envision microplate reader^{10 (link)}.
Firstly, the assay buffer (100 mM Tris-acetate at pH 8.0, 40 mM NaCl, 10 mM Mg-acetate, 0.005% Tween 20 and 1 mM DTT) was added into a 96-well plate. Then, 5 nM recombinant human MTH1, 100 mM dGTP and 0.2U/ml inorganic pyrophosphates were added in sequence. After 60 min reaction, the mixture of 0.45 mg/mL malachite green and 4.2% ammonium molybdate in proportion of 3:1 were added. Then, the plates were incubated and shaken at room temperature for another 60 min. The absorbance was measured at 630 nm using the PerkinElmer Envision micro plate reader.

Zhou W., Ma L., Yang J., Qiao H., Li L., Guo Q., Ma J., Zhao L., Wang J., Jiang G., Wan X., Adam Goscinski M., Ding L., Zheng Y., Li W., Liu H., Suo Z, & Zhao W. (2019). Potent and specific MTH1 inhibitors targeting gastric cancer. Cell Death & Disease, 10(6), 434.

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DPK Modulation of Macrophage Inflammation

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Water-extracted DPK powder was dissolved in incomplete RPMI-1640 culture medium. THP-1 cells were plated in a 96-well plate at a density of 10,000 cells per well and differentiated to macrophages with 20 ng/ml PMA. Following a 24-h exposure, the differentiation medium was replaced with fresh RPMI 1640, and cells were allowed to rest for 24 h. Then, 500 ng/ml of LPS was used to induce inflammation in these macrophages. The cells were treated for 24 h with different concentrations of DPK (1, 10, 100, 300, and 1,000 μg/ml) diluted in media. Cells were pretreated with DPK extract for 24 h before inducing inflammation with LPS. Cell supernatants were collected in a 96-well plate for measuring the levels of pro-inflammatory cytokines: TNF-α, IL-6, and IL-1β. Cytokine levels were measured using ELISA kits (BD Biosciences), according to the manufacturer’s protocol. Absorbance was recorded at 450 nm using an EnVision microplate reader (Perkin Elmer, United States). After washing the cells with PBS, 100 µl alamarBlue (15 μg/ml) was added to each well and incubated for 3 h at 37°C. Subsequently, fluorescence was measured using the EnVision microplate reader (Perkin Elmer, United States) at 530 (excitation) and 590 (emission) nm, and cell viability percentage was calculated.

Balkrishna A., Sakat S.S., Karumuri S., Singh H., Tomer M., Kumar A., Sharma N., Nain P., Haldar S, & Varshney A. (2020). Herbal Decoction Divya-Peedantak-Kwath Alleviates Allodynia and Hyperalgesia in Mice Model of Chemotherapy-Induced Peripheral Neuropathy via Modulation in Cytokine Response. Frontiers in Pharmacology, 11, 566490.

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Cytosafety and Anti-inflammatory Effects of PN-G

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Cytosafety and anti-inflammatory effect of PN-G was evaluated in THP-1 cells, as described earlier (Balkrishna et al., 2020 (link)). Cells, seeded at a density of 5 × 10⁴ cells/well, were differentiated into macrophages with 20 ng/ml with PMA for 16 h before treating with different concentrations (1, 3, 10 and 30 μg/ml) of PN-G for another 24 h. Subsequently, inflammation was induced in these cells with LPS (500 ng/ml) and further co-treated with the above mentioned concentrations of PN-G for the entire duration of inflammation induction of 24 h. Levels of secreted IL-6 and IL-1β in the cell supernatants were measured using ELISA kits according to manufacturer’s protocol. Absorbance was recorded at 450 nm using the Envision microplate reader (Perkin Elmer, United States). Viability of the same cells were determined through Alamar blue staining. After washing the treated cells with PBS, 100 µl Alamar blue (15 μg/ml) was added to each well, and incubated for 3 h at 37°C. Subsequently, fluorescence was measured using Envision microplate reader (Perkin Elmer, United States) at 530 (excitation) and 590 (emission) nm wavelengths.

Balkrishna A., Sinha S., Karumuri S., Srivastava J., Haldar S, & Varshney A. (2022). Peedanil Gold, Herbo-Mineral Formulation, Moderates Cytokine Levels and Attenuates Pathophysiology in Monosodium Iodoacetate Induced Osteoarthritis in SD Rat Model. Frontiers in Pharmacology, 13, 883475.

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GI50 Determination for Cell Viability

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To obtain the GI₅₀ value of a substance, cell viability was measured using MTT (G4000, Promega, USA) for KM12(Luc) cell and Cell titer glo (G7572, Promega, USA) for other cell lines. For adherent cells, cells were seeded at 5,000 cells/well in a 96-well plate and treated with a substance one day after cell seeding. For suspension cells, 10,000 cells/well were seeded and the substance was treated after 4 h stabilization of cells. The substance was treated at 10 points of 1:3 serial dilution (0–100 µmol/L) for 72 h. For MTT assay, 15 µL dye solution was added to each well and incubated 4 h at 37°C. 100 µL of solubilization solution was added and followed 1 hr incubation. Absorbance at 570 nm of each well was recorded using EnVision microplate reader (Perkin Elmer). For Cell titer glo assay, Cell titer glo reagent was treated in each well and measured the luminescence by EnVision microplate reader (Perkin Elmer). Viable cells in each well were normalized by using 0.5% DMSO-treated wells (100%). The inhibitor dose−response curve was fitted and GI₅₀ values were calculated using Prism 7.0 software (GraphPad). All assays were performed in duplicate and the standard deviation (SD) was determined from two independent experiments.

Cho H., Kim N., Murakami T, & Sim T. (2021). Anti-Tumor Activity of AZD4547 Against NTRK1 Fusion Positive Cancer Cells Through Inhibition of NTRKs. Frontiers in Oncology, 11, 757598.

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Quantifying Luciferase Inhibition Screening

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To quantify the rate
of false positives reported during the PbLuc liver stage reconfirmation
screen, hits selected for tertiary round screening (405 compounds
from fresh DMSO stocks) were also tested for luminescent interference
between recombinant firefly luciferase and luciferin. Initially, 40
nL of selected compounds was dispensed using a Gen 4 Plus Acoustic
Transfer System (Biosero, San Diego, USA) at 1:3 in 12-point dose
response format (25 to 141.13 × 10^–6 μM;
final DMSO concentration of 0.5% per well) in 1536-well, white, opaque-bottom
plates (ref# 789173-F, Greiner Bio-One). A 24-point single concentration
series of Luciferase Inhibitor-II (9.8 μM per well) was dispensed
as a positive control, while 96 wells of DMSO at 0.5% per well acted
as the negative control. Separately, a solution of 20 pM recombinant
luciferase (Promega cat# E170A) was prepared on ice by successive
dilutions (first 1:999 then into final solution at 20 pM) in phenol
naive DMEM (Invitrogen cat# 31053-028), 5% FBS (Corning), and 5×
Pen Strep Glutamine (Invitrogen). This solution was dispensed at 8
μL per well into the previously spotted plates using a MultiFlo
dispenser (BioTek) and incubated at room temperature for 1 h prior
to the addition of 1 μL of BrightGlo (Promega). Immediately
after the addition of BrightGlo, plates were read using the EnVision
Microplate reader (PerkinElmer).

Abraham M., Gagaring K., Martino M.L., Vanaerschot M., Plouffe D.M., Calla J., Godinez-Macias K.P., Du A.Y., Wree M., Antonova-Koch Y., Eribez K., Luth M.R., Ottilie S., Fidock D.A., McNamara C.W, & Winzeler E.A. (2020). Probing the Open Global Health Chemical Diversity Library for Multistage-Active Starting Points for Next-Generation Antimalarials. ACS Infectious Diseases, 6(4), 613-628.

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Evaluating Coronil's Impact on A549 Cell Viability

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A549 cell viability was measured using Alamar blue reagent (Hi Media Laboratories, Mumbai, India). Briefly, A549 cells (3 × 10⁴ cells/well) were seeded in 96-well plates and treated for 24 h with various concentrations of Coronil (1–1000 µg/mL). Two hours before termination, 10 µL of Alamar blue (0.15 mg/mL) was added to each well. Cytotoxicity was measured by reading fluorescence at Ex. 540/Em. 590 using the Envision microplate reader (Perkin Elmer, Waltham, MA, USA).

Balkrishna A., Solleti S.K., Verma S, & Varshney A. (2020). Application of Humanized Zebrafish Model in the Suppression of SARS-CoV-2 Spike Protein Induced Pathology by Tri-Herbal Medicine Coronil via Cytokine Modulation. Molecules, 25(21), 5091.

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THP-1 Cell Nitric Oxide Assay

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THP-1 cells were seeded in 96-well culture plates at a density of 2 × 10⁵ cells/ml. Cells were treated with different concentrations of SBKT oil emulsion made in incomplete RPMI-1640 media and incubated for 1 h. Cells were stimulated with LPS (500 ng/ml) and incubated for an additional 24 h at 37ºC in CO₂ incubator. The reactive nitrogen species (RNS) release in the culture media was determined using modified Griess reagent (Sigma), following the manufacturer’s protocol. Absorbance was recorded at 540 nm using Envision Microplate reader (PerkinElmer).

Balkrishna A., Sakat S.S., Joshi K., Joshi K., Sharma V., Ranjan R., Bhattacharya K, & Varshney A. (2019). Cytokines Driven Anti-Inflammatory and Anti-Psoriasis Like Efficacies of Nutraceutical Sea Buckthorn (Hippophae rhamnoides) Oil. Frontiers in Pharmacology, 10, 1186.

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Modulation of Inflammatory Cytokines by SBKT Oil

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THP-1 cells were seeded in 24-well culture plates at a density of 5 × 10⁵ cells/well. For the experiment, SBKT oil was prepared as an emulsion and mixed with the cell culture media at different concentrations: 1.25, 2.5, and 5 μl/ml. THP-1 cells were pre-incubated with the SBKT containing media for 1 h before addition of 1 µg/ml (final concentration) LPS. No LPS was added to the negative control cells. Cell culture supernatants were collected after 24 h, and different pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were measured using ELISA kits (BD Biosciences) following the manufacturer’s protocol. Absorbance was recorded at 450 nm using the Envision microplate reader (PerkinElmer).

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High-throughput Screening for Circadian Modulators

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The chemical screen for circadian clock modulators was conducted at the Chemical Genomics Core facility at the UTHSC-H. The in-house chemical library we screened consists of compounds from NIH Clinical Collection, NCI collection and Microsource Spectrum Collection. The screening was conducted largely based on the protocol previously described (Chen et al., 2012 (link)). Briefly, immortalized fibroblast cells from Clock^Δ19^/+ heterozygous mice expressing the PER2::Luc bioluminescence reporter were plated into 96-well plates. Upon confluency, cells were incubated with 5 μM forskolin for 1–2 h followed by the addition of chemical compounds to the plates with robotic arms (Beckman), and then subjected to continuous monitoring over several days in a temperature-controlled EnVision microplate reader (Perkin Elmer). Data analysis was carried out by using the MultiCycle software (Actimetrics) for measurement of period, phase, and amplitude.

He B., Nohara K., Park N., Park Y.S., Guillory B., Zhao Z., Garcia J.M., Koike N., Lee C.C., Takahashi J.S., Yoo S.H, & Chen Z. (2016). The small molecule Nobiletin targets the molecular oscillator to enhance circadian rhythms and protect against metabolic syndrome. Cell metabolism, 23(4), 610-621.

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Evaluating Withanone's Inhibition of ACE2-RBD Interaction

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The effect of withanone on ACE2-RBD interaction was evaluated using the SARS-CoV-2 inhibitor screening kit (ACRObiosystems, Newark, DE, USA) according to the manufacturer’s protocol. Briefly, wells of the ELISA plate were coated with recombinant truncated SARS-CoV-2 S-protein expressing only RBD. Subsequently, biotinylated human ACE2 protein was allowed to bind to the immobilized RBD in the ELISA wells in the presence of 47 pg/mL (10 pM)–4.7 µg/mL (10 µM) and absence of commercially procured withanone (Sigma, USA). Efficiency of ACE2 binding to RBD was detected through HRP conjugated streptavidin by measuring the absorbance at 450 nm in EnVision microplate reader (Perkin Elmer Inc, Waltham, MA, USA). The absorbance was used to calculate percent inhibition of ACE2-RBD interaction, relative to the reaction mix without withanone. Reaction mix treated with a known inhibitor (provided with the kit) served as a positive control. The observation was represented as a dose-response curve and IC₅₀ calculated using an option built into the GraphPad Prism version 7.0.0 for Windows (GraphPad Software, San Diego, California, USA).

Balkrishna A., Pokhrel S., Singh H., Joshi M., Mulay V.P., Haldar S, & Varshney A. (2021). Withanone from Withania somnifera Attenuates SARS-CoV-2 RBD and Host ACE2 Interactions to Rescue Spike Protein Induced Pathologies in Humanized Zebrafish Model. Drug Design, Development and Therapy, 15, 1111-1133.

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