Fluoroskan ascent fl fluorescence plate reader

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Fluoroskan Ascent FL is a fluorescence plate reader designed for a variety of applications in life science research. It measures fluorescence intensity in microplates, supporting multiple excitation and emission wavelengths. The instrument provides accurate and reliable data for researchers working with fluorescent-based assays.

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Lab products found in correlation

Pnk buffer, by New England Biolabs (1 mentions) Kinase glo, by Promega (1 mentions) Cytoselect tumor transendothelial migration assay kit, by Cell Biolabs (1 mentions) Alamarblue assay, by Thermo Fisher Scientific (1 mentions)

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6 protocols using fluoroskan ascent fl fluorescence plate reader

Evaluating Inhibitor Specificity on MTH1

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Example 2

Background: MTH1 is a DNA repair enzyme with similar properties of OGG1. An experiment to measure the effect of inhibitors in accordance with certain embodiments against MTH1 demonstrates the specificity for OGG1 of inhibitors in accordance with certain embodiments.

Methods: Compound 41 and MTH1 (500 nM) were incubated in 21.8 μL reaction volume PNK buffer (1×) solution of ARGO probe (ATP-linked chimeric nucleotide) (40 μM) at 30° C. (30 min). After that, 5 μL of this reaction solution was mixed with 20 μL of Kinase-Glo. Immediately, luminescence was measured on a Thermo Fluoroskan Ascent FL fluorescence plate reader. (See, e.g., Ji, D., et al., J. Am. Chem. Soc. 2016, 138, 9005-9008, the disclosure of which is incorporated herein by reference.) The slope of initial rate (6 min) was calculated, and % of control was used for inhibition activity. MTH1 was purchased from Abcam, PNK buffer was purchased from New England Biolabs, and Kinase-Glo was purchased from Promega.

Results: As illustrated in FIG. 11, MTH1 possessed >80% enzyme activity in the presence of the inhibitor, indicating that the inhibitor has minimal inhibitory effect on MTH1.

Conclusion: Compound 41 shows low inhibitory effect on MTH1.

US11440887B2. Substituted 1,2,3,4-tetrahydroquinolines as inhibitors of repair enzyme 8-oxoguanine deoxyribonucleic acid glycosylase activity (2022-09-13). The Board of Trustees of the Leland Stanford Junior University [US]. Inventors: Eric T. Kool [US], Yuki Tahara [US].

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OGG1 Knockout MEF Cell Extract Assay

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Ogg1 −/− MEF cells were provided by Dr. Istvan Boldogh (University of Texas Medical Branch at Galveston). ^{[47 (link)]} Ogg1 −/− and WT MEF cells were grown in 3:1 DMEM:F12 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37 °C with 5% CO₂. To obtain cell extract, the protocol by Folco et al. ^{[78 ]} was followed with a few changes: The cells were collected by trypsinization at room temperature instead of scraping. Roche complete mini EDTA-free tablets were used instead of PMSF. The cells were lysed by passing 10-15 times through a 21 gauge needle and 5-10 times through a 16 gauge needle instead of through a Dounce homogenizer. Total protein concentration was determined by the Bradford assay.
Equal amounts (6.7 ng/uL) of WT and Ogg1 −/− MEF cell extract were incubated with 2 μM probe pOGR1 and an additional 10 mM MgCl₂ at 37 °C in 80 μL reaction volumes in a black 384-well plate. Fluorescence was measured at 460 nm following excitation at 355 nm on a Thermo Fluoroskan Ascent FL fluorescence plate reader.

Edwards S.K., Ono T., Wang S., Jiang W., Franzini R.M., Jung J.W., Chan K.M, & Kool E.T. (2015). In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage. Chembiochem : a European journal of chemical biology, 16(11), 1637-1646.

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Investigating OGG1 Inhibitor Mechanism

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Example 7

Background: K_mand K_catare measures of enzyme activity and can change in the presence of inhibitory molecules. A study was performed to measure these changes to determine the activity of some embodiments of OGG1 inhibitors to determine whether these inhibitors exhibit competitive, uncompetitive, or noncompetitive inhibition of OGG1.

Methods: For Michaelis-Menten curves: Inhibitor 41 and hOGG1 (100 nM) were incubated in NEBuffer 4 (1×) with BSA (1×) at 37° C. (15 min) in 100 μL reaction volumes in a black 96-well plate. After that, OGR1 (0.8-10 μM) was added to the reaction mixture. Fluorescence at 460 nm was measured on a Thermo Fluoroskan Ascent FL fluorescence plate reader (Aex=355 nm). The initial rate (12 min) was calculated. Lineweaver-Burk plot was leaded from the Michaelis-Menten curves. K_mand V_maxwere calculated from the equation. K_catwas calculated from K_cat=V_max/[enzyme].

Results: As illustrated in FIG. 9 and summarized in Table 7, the effect of the inhibitor on OGG1 had little to no effect on K_mand a lowering of K_cat.

Conclusion: This study indicates that compound 41 is possibly a noncompetitive inhibitor against OGG1.

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Transendothelial Migration Assay for Tumor Cells

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Transendothelial migration assay was performed using the CytoSelect™ Tumor Transendothelial Migration Assay kit (Cell Biolabs). Briefly, HLECs (1 × 10⁵ cells) were cultured on porous inserts (8 μm) in 24-well plates for 72 h. SAS-L1 cells transfected with EFNB2 or control scrambled siRNA were labeled with CytoTracker™ and placed over the HLEC monolayer in the presence of the clustered Fc. After 24 h, non-migrated cells in the inner chamber were removed with cotton-tipped swabs. The cells that migrated outside the inner chamber were collected and resolved in lysis buffer. Fluorescence intensity was measured with a Fluoroskan Ascent FL fluorescence plate reader (Thermo Lab systems, Helsinki, Finland) at excitation and emission wavelengths of 485 and 520 nm, respectively.

Sasabe E., Tomomura A., Tomita R., Sento S., Kitamura N, & Yamamoto T. (2017). Ephrin-B2 reverse signaling regulates progression and lymph node metastasis of oral squamous cell carcinoma. PLoS ONE, 12(11), e0188965.

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Amyloid-beta Fibrillation Assay

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To assess Aβ_1–42 fibril formation, 200 μL of each incubation sample was mixed with 0.5 μL of 10 mM ThT (Sigma Aldrich, Shanghai, China) in a Costar 96-well plate with black/clear bottoms. The plate was incubated at 37 °C with shaking at 120 rpm. ThT fluorescence intensity was measured at each time point using a Thermo Scientific (Madison, WI, USA) Fluoroskan Ascent FL fluorescence plate reader set to 440 nm excitation and 485 nm emission. Data acquisition and analysis were performed using Ascent Software for Fluoroskan Ascent software ver 2.6.

Zhao X., Mou C., Xu J., Cui W., Shi Y., Wang Y., Luo T., Guo W., Ye J, & Chen W. (2024). Protection of Si Nanowires against Aβ Toxicity by the Inhibition of Aβ Aggregation. Molecules, 29(9), 1980.

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Alamar Blue™ Assay for Cell Metabolism

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In order to assess cell metabolic activity Alamar Blue™ assay (Invitrogen, Paisley, UK) was used. Cells seeded onto tissue culture plastic served as a positive control and media only wells provided a negative control. Cells were incubated at 37 o C at 5% CO2 in air and cell culture media was replenished on days 0, 1, 3 and 6. Cell metabolism was assessed using Alamar Blue™ assay according to manufacturer's guidelines on days 1, 3 and 7. Media from the wells was removed and fresh media containing 10% Alamar Blue™ solution was added to each well. Following a 4 hr incubation wrapped in aluminium foil, 100l of the media from each well was placed into a 96-well plate and analysed using a fluorescent plate reader (Fluoroskan Ascent FL™ Fluorescence Plate Reader, ThermoScientific, USA) at an excitation and emission wavelengths of 530nm and 620nm (n=6).

Magill L.J., Tanska A., Keshtgar M., Mosahebi A, & Jell G. (2019). Mechanical and surface chemical analysis of retrieved breast implants from a single centre. Journal of the mechanical behavior of biomedical materials, 91.

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