Enspire alpha plate reader

Manufactured by PerkinElmer

Sourced in United States

The EnSpire Alpha Plate Reader is a high-performance microplate reader designed for a wide range of applications in life science research and drug discovery. It features advanced detection capabilities, including simultaneous multi-label detection and fluorescence intensity, time-resolved fluorescence, and luminescence measurements. The EnSpire Alpha Plate Reader provides accurate and reliable data to support scientific investigations.

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Lab products found in correlation

Glucose assay kit, by Merck Group (3 mentions) Dual luciferase reporter assay kit, by Promega (2 mentions) Aspergillus niger amyloglucosidase, by Merck Group (2 mentions) S adenosylmethionine, by Merck Group (1 mentions) Alphascreen streptavidin donor beads, by PerkinElmer (1 mentions) Immunoassay buffer, by PerkinElmer (1 mentions) Veriti thermal cycler, by Thermo Fisher Scientific (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions) Cyquant direct cell proliferation assay, by Thermo Fisher Scientific (1 mentions) Insulin human alphalisa detection kit, by PerkinElmer (1 mentions) Proinsulin elisa kit, by Mercodia (1 mentions) Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions) Acceptor beads, by PerkinElmer (1 mentions) Proclin 300, by Merck Group (1 mentions) Biotin n hydroxysuccinimide ester, by Merck Group (1 mentions) 4 methylumbelliferyl α d glucopyranoside, by Merck Group (1 mentions) Bodipy, by PerkinElmer (1 mentions) Bodipy ldl, by Thermo Fisher Scientific (1 mentions) Cyquant cell proliferation assay, by Thermo Fisher Scientific (1 mentions) Dii ldl, by Thermo Fisher Scientific (1 mentions)

20 protocols using enspire alpha plate reader

Measuring Glycogen and GAA Activity

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GAA activity was measured as previously described.⁸¹ (link) Briefly, 10 μL of sample, either tissue homogenate or plasma, was incubated for 1 h at 37°C with 20 μL of 4-methylumbelliferyl α-d-glucopyranoside (3 mM), diluted in acetate buffer solution (pH 4.65) (Sigma-Aldrich). The reaction was stopped using the carbonate solution, and the fluorescence (λ_ex 360 nm/λ_em 449 nm) was read with an EnSpire alpha plate reader (PerkinElmer, Waltham, MA). A standard curve was prepared using 4-methylumbelliferone, diluted in 0.5 M of carbonate solution (pH 10.5).
Glycogen content was measured indirectly in tissue homogenates as the glucose released after total digestion with Aspergillus niger amyloglucosidase (Sigma-Aldrich, St. Louis, MO, USA). Samples were incubated for 5 min at 95°C and then cooled at 4°C; 25 μL of amyloglucosidase diluted 1:50 in 0.1 M potassium acetate (pH 5.5) was then added to each sample. A control reaction without amyloglucosidase was prepared for each sample. Both sample and control reactions were incubated at 37°C for 90 min. The reaction was stopped by incubating samples for 5 min at 95°C. The glucose released was determined using a glucose assay kit (Sigma-Aldrich, St. Louis, MO, USA) and by measuring resulting absorbance at the EnSpire alpha plate reader (PerkinElmer, Waltham, MA, USA) at 540 nm.

Cagin U., Puzzo F., Gomez M.J., Moya-Nilges M., Sellier P., Abad C., Van Wittenberghe L., Daniele N., Guerchet N., Gjata B., Collaud F., Charles S., Sola M.S., Boyer O., Krijnse-Locker J., Ronzitti G., Colella P, & Mingozzi F. (2020). Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase. Molecular Therapy, 28(9), 2056-2072.

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Indirect Glycogen Quantification Assay

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Glycogen content was measured indirectly in tissue homogenates as the glucose released after total digestion with Aspergillus Niger amyloglucosidase (diastase, Sigma-Aldrich, Saint Louis, MO). Samples were incubated for 5 min at 95 °C and then cooled at 4 °C; 25 μl of amyloglucosidase diluted 1:50 in 0.1 M potassium acetate pH 5.5 were then added to each sample. A control reaction without amyloglucosidase was prepared for each sample. Both sample and control reactions were incubated at 37 °C for 90 min. The reaction was stopped by incubating samples for 5 min at 95 °C. The glucose released was by a glucose assay kit (Sigma-Aldrich, Saint Louis, MO) and the resulting absorbance was acquired on an EnSpire alpha plate reader (Perkin-Elmer, Waltham, MA) at a wavelength of 540 nm.

Laforêt P., Inoue M., Goillot E., Lefeuvre C., Cagin U., Streichenberger N., Leonard-Louis S., Brochier G., Madelaine A., Labasse C., Hedberg-Oldfors C., Krag T., Jauze L., Fabregue J., Labrune P., Milisenda J., Nadaj-Pakleza A., Sacconi S., Mingozzi F., Ronzitti G., Petit F., Schoser B., Oldfors A., Vissing J., Romero N.B., Nishino I, & Malfatti E. (2019). Deep morphological analysis of muscle biopsies from type III glycogenesis (GSDIII), debranching enzyme deficiency, revealed stereotyped vacuolar myopathy and autophagy impairment. Acta Neuropathologica Communications, 7, 167.

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Measuring cAMP Levels in Transfected Cells

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HEK 293 cells were seeded at a density of 15,000 cells per well in a 96 well plate and left to attach overnight. The next day, cells were transfected with 50 ng of plasmidic DNA (pcDNA3.1, WT GPR3-pcDNA and mutants, either with or without barr2-pEGFP) as described above. Forty eight hours after transfection, cells were incubated with 100 μl of HHBS containing 1 mM IBMX (phosphodiesterase inhibitor) at 37 °C and for 30 minutes and stimulated with 1 μL of DPI (or forskolin) for additional 30 minutes. DPI and forskolin were dissolved in DMSO at different concentrations, according to the experimental design. The medium was removed and 50 μl of ice cold 100% ethanol was added to each well. After evaporation, cells were resuspended in 50 μl of Lysis buffer (0.3% Tween 20 in HHBS buffer) and incubated at room temperature for 10 minutes. The cAMP concentration in the cell lysate (5 μL) was determined with a cAMP AlphaScreen Assay kit (Perkin Elmer) and a EnSpire Alpha Plate Reader (Perkin Elmer) according to the manufacturer’s instructions.

Capaldi S., Suku E., Antolini M., Di Giacobbe M., Giorgetti A, & Buffelli M. (2018). Allosteric sodium binding cavity in GPR3: a novel player in modulation of Aβ production. Scientific Reports, 8, 11102.

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Histone H3 Methyltransferase Assay by ALPHA

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An amplified luminescence proximity homogeneous assay (ALPHA) was performed with a modified version of a previously described method^{24 (link)}. Recombinant G9a proteins (BPS Bioscience) were incubated with 15 μM S-adenosyl-methionine (Sigma) and 50 nM biotinylated histone H3 (1-21; AnaSpec) in the assay buffer (50 mM Tris-HCl (pH 9.0), 50 mM NaCl, 0.01% Tween-20, and 1 mM DTT). After incubation at room temperature for 1 h, AlphaLISA anti-H3K9me2 acceptor beads (final concentration 10 μg/mL, PerkinElmer, AL117C) and AlphaScreen streptavidin donor beads (final concentration 10 μg/mL, PerkinElmer, 6760002B) were added and incubated for an additional 1 h prior to detection of the alpha signal with an EnSpire Alpha plate reader (PerkinElmer). ALPHA profile was analyzed using Origin (OriginLab).

Takase S., Hiroyama T., Shirai F., Maemoto Y., Nakata A., Arata M., Matsuoka S., Sonoda T., Niwa H., Sato S., Umehara T., Shirouzu M., Nishigaya Y., Sumiya T., Hashimoto N., Namie R., Usui M., Ohishi T., Ohba S.I., Kawada M., Hayashi Y., Harada H., Yamaguchi T., Shinkai Y., Nakamura Y., Yoshida M, & Ito A. (2023). A specific G9a inhibitor unveils BGLT3 lncRNA as a universal mediator of chemically induced fetal globin gene expression. Nature Communications, 14, 23.

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Heat Shock Effect on B-Raf Inhibition

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A375 cells were harvested to a density of 2 × 10⁷ cells/mL in Hanks’ balance salt solution (HBSS) and treated with 10 µM dabrafenib or 0.1% DMSO. Cells were incubated for 1 h under tissue culture conditions with gentle continuous rotation. Cell suspensions were aliquoted into PCR strips (30 µL/tube) and each strip subjected to a 3 min heatshock at the indicated temperature using a Veriti thermal cycler (Thermo Scientific, Waltham, MA). Samples were stored on ice and lysed by three repetitive freeze–thaws in liquid nitrogen. Five microliters of lysate was transferred to a 96-well plate and anti-B-Raf antibodies Abnova (Taipei, Taiwan) H00000673-M02 and SantaCruz (Dallas, TX) sc-9002, prepared in ImmunoAssay Buffer (PerkinElmer, AL000F), were added to a final concentration of 0.3 nM in 25 µL volume. Following 1 h incubation, AlphaScreen signal was developed by addition of Anti-Mouse IgG Alpha Donor Beads to a final concentration of 80 µg/mL, and Anti-Rabbit IgG AlphaLISA Acceptor Beads to a final concentration of 20 µg/mL. After 16 h, plates were analyzed using an EnSpire Alpha Plate Reader (PerkinElmer).

Shaw J., Dale I., Hemsley P., Leach L., Dekki N., Orme J.P., Talbot V., Narvaez A.J., Bista M., Martinez Molina D., Dabrowski M., Main M.J, & Gianni D. (2018). Positioning High-Throughput CETSA in Early Drug Discovery through Screening against B-Raf and PARP1. Slas Discovery, 24(2), 121-132.

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Quantifying ACVR1 Kinase Activity via pSmad1 Assay

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To indirectly measure kinase activity of ACVR1 receptors, we used the AlphaScreenSureFire Smad1 p-Ser463/p465 assay (PerkinElmer, catalog # TGRSM1S500). The assay is based on the formation of sandwich antibody complex (comprised of a donor bead recognizing Smad1 and an acceptor bead, recognizing Smad1, when phosphorylated at Ser463/465) that only forms, when both beads are in close proximity to allow energy transfer after excitation with light at 680nm. Emission was captured at 520–620 nm using an EnSpire Alpha Plate Reader (PerkinElmer). Immortalized MEFs were plated in 24-well plates (4.5×10⁴ cells/well) and incubated at 37°C overnight. Cells were transfected as described above (cell transfection) with indicated ACVR1 receptor expression plasmid and renilla luciferase normalization reporter. The next day cells were starved for 2 hours in serum-reduced media (0.5% FBS in DMEM) followed by stimulation with ligand for 1 hour. Cells were then lysed as described above. Detection of phosphorylated Smad1 was done following manufacturer’s instruction. Renilla activity was detected as described in previous section (Luciferase reporter gene assay). Relative pSmad1 level was calculated by normalization to renilla activity and to value of cells expressing the WT ACVR1 receptor under untreated condition.

Haupt J., Xu M, & Shore E.M. (2017). Variable signaling activity by FOP ACVR1 mutations. Bone, 109, 232-240.

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Cytotoxic Effect of PG on Oral Cancer Cells

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Cytotoxicity was measured by MTT assay. 7 × 10³ cells per well of SAS/OECM1 were seeded in 96-well plates and incubated at 37 °C, 5% CO₂ overnight. Then, the cells were treated with various concentrations of PG (0.1, 0.5, 1.0, and 5.0 μM) for 24 h. After treatment, 20 μL per well of 50 mg/mL MTT (Thermo-Fisher) solution was added and incubated at 37 °C for 3 h. As incubation finished, all liquid in wells were replaced to dimethyl sulfoxide and the absorbance at 570 nm was measured by EnSpire Alpha plate reader (Perkin Elmer, Waltham, MA, USA). The absorbance at 570 nm was positively correlated to the number of viable cells so the cell viability was represented as the percentage of absorbance at 570 nm between treated and untreated cells.

Cheng M.F., Lin C.S., Chen Y.H., Sung P.J., Lin S.R., Tong Y.W, & Weng C.F. (2017). Inhibitory Growth of Oral Squamous Cell Carcinoma Cancer via Bacterial Prodigiosin. Marine Drugs, 15(7), 224.

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Quantifying Glucose-Stimulated Insulin Secretion

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Silenced EndoC-βH1 cells were starved overnight at 48 h post-transfection in culture medium containing 2.8 M glucose followed by 30 min incubation in 0 mM glucose. Insulin secretion was initiated through static incubations in the indicated glucose or secretagogues conditions for 1 h. Insulin containing supernatant was collected, and cells were lysed in ice-cold acid ethanol to release intracellular insulin. Insulin was quantified using the Insulin (human) AlphaLISA Detection kit and the EnSpire Alpha Plate Reader (both Perkin Elmer) based on 1:10 and 1:200 dilutions for supernatant and insulin content, respectively. Intracellular proinsulin was quantified using the Proinsulin ELISA kit (Mercodia). Secreted insulin was normalized to the level of intracellular insulin content or cell count, which was measured before cell lysis using the CyQUANT direct cell proliferation assay (Invitrogen).

Rottner A.K., Ye Y., Navarro-Guerrero E., Rajesh V., Pollner A., Bevacqua R.J., Yang J., Spigelman A.F., Baronio R., Bautista A., Thomsen S.K., Lyon J., Nawaz S., Smith N., Wesolowska-Andersen A., Fox J.E., Sun H., Kim S.K., Ebner D., MacDonald P.E, & Gloyn A.L. (2022). A genome-wide CRISPR screen identifies CALCOCO2 as a regulator of beta cell function influencing type 2 diabetes risk. Nature Genetics, 55(1), 54-65.

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Measuring Wnt Signaling in T-ALL Cells

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β-catenin luciferase reporter plasmids were obtained from Addgene (cat. #12457 and #12456). 3 µg of TOP-FLASH or FOP-FLASH plasmids were transfected onto 1 × 10⁶ human T-ALL cells cultured in single wells of a 6-well plate using Lipofectamine LTX (Thermo Fisher; cat. #15,338,100). Luciferase activity was measured 24 h after transfection using a Dual-Luciferase reporter assay kit (Promega; cat. #E1910) on an Enspire alpha plate reader (Perkin Elmer, Waltham, MA).

Tan Y., Sementino E., Liu Z., Cai K.Q, & Testa J.R. (2020). Wnt signaling mediates oncogenic synergy between Akt and Dlx5 in T-cell lymphomagenesis by enhancing cholesterol synthesis. Scientific Reports, 10, 15837.

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Glycogen Quantification in Mouse Tissues

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Homogenates of mouse tissues were prepared as indicated for GAA activity. Glycogen content was measured indirectly in tissue homogenates as glucose released after total digestion with Aspergillus niger amyloglucosidase (Sigma-Aldrich), as reported previously.⁴⁷ Samples were incubated for 5 min at 95°C and then cooled at 4°C; 25 μL of amyloglucosidase diluted 1:50 in 0.1 M potassium acetate (pH 5.5) was then added to each sample. A control reaction without amyloglucosidase was prepared for each sample. Both digestion and control samples were incubated at 37°C for 90 min and then at 95°C for 5 min. The glucose released was determined using a colorimetric assay (glucose assay kit, Sigma-Aldrich) and by measuring the absorbance at 540 nm using an EnSpire Alpha plate reader (PerkinElmer).

Colella P., Sellier P., Costa Verdera H., Puzzo F., van Wittenberghe L., Guerchet N., Daniele N., Gjata B., Marmier S., Charles S., Simon Sola M., Ragone I., Leborgne C., Collaud F, & Mingozzi F. (2018). AAV Gene Transfer with Tandem Promoter Design Prevents Anti-transgene Immunity and Provides Persistent Efficacy in Neonate Pompe Mice. Molecular Therapy. Methods & Clinical Development, 12, 85-101.

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