Enspire plate reader

Manufactured by PerkinElmer

Sourced in United States

The EnSpire plate reader is a multi-mode detection platform that provides sensitive and accurate quantification of fluorescence, luminescence, and absorbance in microplates. It offers a range of detection modes to support diverse assay types and sample types.

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Lab products found in correlation

Luciferase assay system, by Promega (8 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions) Optiplate 96, by PerkinElmer (5 mentions) Celltiter glo reagent, by Promega (4 mentions) Prism 8, by GraphPad (4 mentions) Bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Nanodrop, by Thermo Fisher Scientific (4 mentions) Celltiter glo, by Promega (3 mentions) Celltiter glo assay, by Promega (3 mentions) Alphascreen protein a acceptor beads, by PerkinElmer (3 mentions) Steadyglo reagent, by Promega (3 mentions) Quanti blue reagent, by InvivoGen (3 mentions) Dual luciferase reporter assay system, by Promega (3 mentions) Resazurin sodium salt, by Merck Group (2 mentions) Pierce gaussia luciferase flash assay kit, by Thermo Fisher Scientific (2 mentions) Lance ultra camp kit, by PerkinElmer (2 mentions) Janus automatic pipetting workstation, by PerkinElmer (2 mentions) 2100 bioanalyzer, by Thermo Fisher Scientific (2 mentions) Kapa hotstart hifi 2 readymix, by Roche (2 mentions) Nextera xt library prep system, by Illumina (2 mentions)

184 protocols using enspire plate reader

Cell Proliferation and ALP Activity

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Cell proliferation was assessed based on DNA quantification at four different time points (day 0, day 1, day 7 and day 28) according to previously published protocols [30] . Day 0 refers to an aliquot of the initial cell seeding suspension (250,000 cells, see 2.2.1).
Briefly, constructs were washed in 1x PBS, and transferred to 1.5 mL Eppendorf tubes. Cells were lysed in 0.1 v/v% triton-X and repeated freeze thawing cycles. DNA was quantified with Hoechst and based on a standard curve of calf thymus DNA (Sigma). Fluorescence was measured at 360 nm excitation and 460 nm emission wavelengths (Perkin Elmer EnSpire Plate Reader, Waltham, MA, USA). ALP activity on day 0 aliquots, and on day 1, day 7 and day 28 constructs was quantified based on the dephosphorylation of p-nitrophenyl phosphate (pNPP, Sigma) by ALP. Cell lysate was incubated with pNPP at 37 °C for 2 h. Serial dilutions of paranitrophenol served as a standard curve for quantification. Absorbance was measured at 405 nm (Perkin Elmer EnSpire Plate Reader, Waltham, MA, USA). Enzymatic activity was then calculated based on the conversion rate of 1 U = 1 µmol pNPP per min.

Guex A.G., Puetzer J.L., Armgarth A., Littmann E., Stavrinidou E., Giannelis E.P., Malliaras G.G, & Stevens M.M. (2017). Highly porous scaffolds of PEDOT:PSS for bone tissue engineering. Acta Biomaterialia, 62, 91-101.

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EGFP Fluorescence Screening Protocol

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Compounds were diluted at 100 uM in PBS and the wavelength of maximum absorption at 26 °C was measured on an Enspire plate reader from Perkin Elmer. In order to determine effect of compounds on EGFP fluorescence, 150 ng of recombinant enhanced Green Fluorescent Protein (EGFP) were diluted in PBS and incubated for 4 h at Room temperature with indicated amounts of compounds. EGFP fluorescence was measured on an Enspire plate reader from Perkin Elmer (Ex_480 nm/Em_510 nm)^{38 (link),39}.

Bellón-Echeverría I., Carralot J.P., Del Rosario A.A., Kueng S., Mauser H., Schmid G., Thoma R, & Berger I. (2018). MultiBacMam Bimolecular Fluorescence Complementation (BiFC) tool-kit identifies new small-molecule inhibitors of the CDK5-p25 protein-protein interaction (PPI). Scientific Reports, 8, 5083.

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Doxycycline-Induced Chloroquine Sensitivity

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Doxycycline-inducible cell lines were pre-treated with doxycycline for 24 hours to induce CA, then harvested for drug screening. 1000 cells in 150 μL media were plated per well in 96 well plates. Cells were allowed to attach overnight, then chloroquine was added the next morning in 50 μL. After 5 days of incubation in the presence of chloroquine and doxycycline, 50μL of Cell Titer Glo (Promega) was added to each well, incubated for 15 minutes at room temperature, and luminescence was analyzed using an Enspire Plate Reader (Perkin Elmer) at the UW Small Molecule Screening and Synthesis Facility. From each experimental luminescence value, we subtracted the luminescence value of wells with media plus Cell Titer Glo (indicating background luminescence without cells) and normalized to the luminescence value of untreated cells within each condition. This screen was replicated using Vita-Orange Cell Viability Reagent (Biotool) in 96-well plates (same protocol as above), as well as crystal violet staining in 24-well plates. For crystal violet staining, cells were pre-treated with doxycycline for 24 hours, then harvested and plated in 24-well plates at a density of 5000 cells/well. Chloroquine was added the next day, and cells were incubated for 5 days before staining with crystal violet.

Denu R.A., Kaur G., Sass M.M., Lakkaraju A, & Burkard M.E. (2019). Centrosome amplification in cancer disrupts autophagy and sensitizes to autophagy inhibition. Molecular cancer research : MCR, 18(1), 33-45.

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Intracellular Dose-Response Assay for Mtb

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Intracellular dose-response curves were carried out as previously described (54 (link)). Briefly, a firefly luciferase (FFluc) reporter strain of Mtb CDC1551 (55 (link)) was grown to mid-log phase. Cultured resting primary bone marrow-derived macrophages were isolated from C57Bl/6 mice and seeded into 96 well plates as described (54 (link)). Macrophages were then infected with the reporter strain at a MOI of 1 (54 (link)). Wells were then treated in duplicate with selected compounds in a range of either 80 µM–8.3 nM or 5.12 µM–8.3 nM (8 or 12 dose points, 2.5-fold dilutions). DMSO and 0.3 µM RIF were included as controls. 96 wells plates were then incubated in vented re-sealable bags with a wet paper towel at 37°C with 5.0% CO₂ for 6 days. After 6 days, the Bright-Glo luciferase agent (Promega) was added to the 96 wells plates in a 1:1 ratio for each well. Plates were then read using a PerkinElmer Enspire plate reader. Percent intracellular growth inhibition was calculated relative to DMSO and RIF controls. EC₅₀s, Hillslopes, and EC₉₀s were calculated as described above using Graphpad Prism 8 Software.

Williams J.T., Giletto M., Haiderer E.R., Aleiwi B., Krieger-Burke T., Ellsworth E, & Abramovitch R.B. (2023). The Mycobacterium tuberculosis MmpL3 inhibitor MSU-43085 is active in a mouse model of infection. Microbiology Spectrum, 12(1), e03677-23.

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Quantifying Transfection Efficiency in NSC34 Cells

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NSC34 cells were plated in 24‐well plate at density of 4 × 10⁴ cells/well (6 wells for each condition to be tested; n = 3) and transfected with 0.4 μg of pCMV‐βgal or pSV‐βgal plasmids. After 48 h of transfection, cells were lysed in 250 μl of lysis buffer (Promega) and 100 μl of samples was added to 750 μl of assay buffer (60 mM Na₂HPO₄ 40 mM NaH₂PO₄ 10 mM KCl, 1 mM MgSO₄, pH 7.0) in presence of 4 mg/ml of β‐galactosidase substrate o‐nitrophenyl‐b‐D‐galactopyraniside (ONPG; Sigma‐Aldrich,) and incubated at 37°C until yellow colour appearance. Then, 500 μl of 1 M Na₂CO₃ was added and 200 μl of the final solution was transferred into a 96‐well plate and 420‐nm absorbance was evaluated using Enspire plate‐reader (PerkinElmer).

Licata N.V., Cristofani R., Salomonsson S., Wilson K.M., Kempthorne L., Vaizoglu D., D’Agostino V.G., Pollini D., Loffredo R., Pancher M., Adami V., Bellosta P., Ratti A., Viero G., Quattrone A., Isaacs A.M., Poletti A, & Provenzani A. (2021). C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation. The EMBO Journal, 41(1), e105026.

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Quantifying PGE2 in Cell Supernatants

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Briefly, cultured cell supernatant samples were collected from different groups and centrifuged at 3000 g for 5 min to pellet cell debris. The concentration of PGE2 was determined using enzyme-linked immunosorbent assay (ELISA) kit (HM10090, Bioswamp, Wuhan, China) according to the manufacturer’ instructions. The optical density (OD) was measured with the EnSpire plate reader (PerkinElmer) at 450 nm.

Zhong Z., Liu Z., Zheng R., Chai J, & Jiang S. (2022). miR-132-3p Modulates DUSP9-Dependent p38/JNK Signaling Pathways to Enhance Inflammation in the Amnion Leading to Labor. International Journal of Molecular Sciences, 23(3), 1864.

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Assessing Multidrug Resistance in K562 Cells

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For assessment of drug toxicity, multidrug-resistant and parental K562 cells in duplicates of 10 000 cells/well, were seeded in black 96-well clear bottom polystyrene microplates (Sigma) containing various drug concentrations diluted in RPMI supplemented with 5% heat inactivated FBS. Following, 72 h incubation at 37 °C, 10 μg/ml resazurin sodium salt (Sigma) was added to each well and further incubated for 2 h at 37 °C. Resorufin fluorescence was measured at a wavelength of 590 nm using an Enspire plate reader (Perkin Elmer).

Langebäck A., Bacanu S., Laursen H., Mout L., Seki T., Erkens-Schulze S., Ramos A.D., Berggren A., Cao Y., Hartman J., van Weerden W., Bergh J., Nordlund P, & Lööf S. (2019). CETSA-based target engagement of taxanes as biomarkers for efficacy and resistance. Scientific Reports, 9, 19384.

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Measuring Senescence-Associated β-Galactosidase Activity

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SAβG activity was measured in a cell lysate after adding 50 μl of: citric acid-sodium phosphate buffer (40–60 mM, pH 6.0), 1 mM of X-gal (Gold biotechnology), 0.1 mM of nitro blue tetrazolum salt (NBT, Gold biotechnology) and 0.1% of Triton X100 (Sigma-Aldrich). After 1 h, an ATP detection solution (20 μl, Cell Titer-Glo, Promega) was added to the samples. The reagents were added by MultiFlo FX dispenser (Biotek). Sample SAβG activity was measured at 615 nm with simultaneous detection of luciferase activity as a readout of ATP using an Enspire plate reader (Perkin Elmer). Viability was calculated as a ratio of luminescence values for each compound to corresponding plate-average vehicle-only values after background subtraction. Screening data was analyzed using Pipeline Pilot (Biovia) software package. All readings were averaged and normalized on corresponding controls. The output was a ratio of SAβG activity divided by the level of ATP.

Vatolin S., Radivoyevitch T, & Maciejewski J.P. (2019). New drugs for pharmacological extension of replicative life span in normal and progeroid cells. NPJ Aging and Mechanisms of Disease, 5, 2.

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Quantifying Genomic DNA Methylation

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For total genomic methylation studies, digested gDNA reactions were first diluted tenfold in water. One microlitre (i.e., 200 pg of DNA) was then used in the MBD/HRP assay to estimate levels of methylation. MBD-modified magnetic beads (NEB, USA) were prepared and used as recommended by the manufacturer. To enrich for methylated DNA, the provided 1× MBD buffer was supplemented with 600 mM NaCl. After a 15-min incubation with DNA targets, the MBD beads were isolated with a magnet and the supernatant was removed. The MBD beads were then resuspended in 20 μL of 1/1000 HRP solution (BD Biosciences, Australia) in 1× MBD buffer for 10 mins. The MBD beads were then washed four times with 1× MBD buffer. Finally, 100 μL of 1-Step™ TMB substrate solution (Thermo Scientific, Australia) and colorimetric changes were monitored at 650 nm over 30 min on the EnSpire® plate reader (Perkin Elmer, Australia). Absorbance values at 20 min were used in subsequent data analysis.

Wee E.J., Ngo T.H, & Trau M. (2015). Colorimetric detection of both total genomic and loci-specific DNA methylation from limited DNA inputs. Clinical Epigenetics, 7(1), 65.

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HIV-1 RT Inhibitor Assay Protocol

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The extracts at 1 mg/mL were tested for HIV-1 RT inhibitor using an HIV-1 reverse transcriptase assay, colorimetric kit (Roche, Germany) according to manufacturer’s protocol. Nevirapine (200 μM), a HIV-1 RT inhibitory drug was used as a positive control. DMSO (1%, v/v) was determined as a vehicle control. The color reactions were measured at wavelength 405 nm and 490 nm as a reference wavelength by using an EnSpire plate reader (Perkin-Elmer).

Sillapachaiyaporn C., Rangsinth P., Nilkhet S., Moungkote N, & Chuchawankul S. (2021). HIV-1 Protease and Reverse Transcriptase Inhibitory Activities of Curcuma aeruginosa Roxb. Rhizome Extracts and the Phytochemical Profile Analysis: In Vitro and In Silico Screening. Pharmaceuticals, 14(11), 1115.

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