Ascent software version 2

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, Finland, United States

Ascent software version 2.6 is a data analysis software tool developed by Thermo Fisher Scientific. It provides core functionalities for processing and analyzing data generated from various Thermo Fisher Scientific laboratory instruments.

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ASCENT version 2.6 software Ascent software Ascent 2.6

14 protocols using ascent software version 2

Proliferation of Lung Cancer Cells with Dox-Loaded Nanoparticles

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Cultured cells were detached with a trypsin–ethylenediami-netetraacetic acid (EDTA) solution (0.25%) and seeded into 24-well plates at a density of 15×10³ A549 cells/well and 2×10⁴ LL/2 cells/well. After incubation for 24 hours under culture conditions, the cells were treated with either free Dox (aqueous solution), blank (Dox-unloaded) PBCA NPs, or Dox-loaded PBCA NPs in increasing drug-equivalent concentrations from 0.01 to 10 μM. After an incubation time of 48 hours, a modified sulforhodamine B (SRB) protocol was followed.25 (link) The cells were washed three times with PBS, and 300 μL of 10% trichloroacetic acid was used to fix the cells (20 minutes at 4°C). Then, the cells were washed three times with distilled water and left to dry before 300 μL of SRB was added again. Incubation under mechanical stirring was continued for 20 minutes, and the excess SRB was removed by the addition of a 1% acetic acid solution. Then, the cells were left to dry. Finally, 200 μL of Trizma^® (10 mM, pH 10.5; Sigma-Aldrich) was used for dye resuspension. The optical density (OD) of SRB was measured at 492 nm (Titertek^® Multiskan colorimeter; Flow Laboratories Ltd, Oldham, UK) and analyzed with Ascent software version 2.6 (Thermo Labsystems, Helsinki, Finland).
The percentage of relative proliferation (RP, %) was calculated:

RP (%) = \frac{OD of treated cells}{OD of untreated cells} \times 100

Melguizo C., Cabeza L., Prados J., Ortiz R., Caba O., Rama A.R., Delgado Á.V, & Arias J.L. (2015). Enhanced antitumoral activity of doxorubicin against lung cancer cells using biodegradable poly(butylcyanoacrylate) nanoparticles. Drug Design, Development and Therapy, 9, 6433-6444.

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Antioxidant Activity Determination by PSC Assay

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The antioxidant activity of extracts was determined by using a PSC assay developed in our lab [24] (link) with modifications [25] . Just prior to use in the reaction, 107 µL of 2.48 mM dichlorofluorescein diacetate was hydrolyzed to dichlorofluorescein with 893 µL of 1.0 mM KOH for 5 min in a vial to remove the diacetate moiety and then diluted with 7 mL of 75 mM phosphate buffer (pH 7.4). ABAP (200 mM) was prepared fresh in the buffer and was kept at 4°C between runs. In an assay, 100 µL of extracts was diluted in 75 mM phosphate buffer (pH 7.4) and then transferred into reaction cells on a 96-well plate, and 100 µL of dichlorofluorescein was added. The 96-well plate was loaded into a Fluoroskan Ascent fluorescence spectrophotometer (Thermo Labsystems, Franklin, MA), and the solution in each cell was mixed by shaking at 1200 rpm for 20 s. The reaction was then initiated by adding 50 µL of ABAP from the autodispenser on the equipment. Each set of dilutions for a replicate and control was analyzed three times in adjacent columns. The reaction was carried out at 37°C, and fluorescence was monitored at 485 nm excitation and 538 nm emission with the fluorescence spectrophotometer. The buffer was used for control reactions. Data were acquired with Ascent software, version 2.6 (Thermo Labsystems). Data were reported as mean ± SD with three replicates.

Liang Z., Cheng L., Zhong G.Y, & Liu R.H. (2014). Antioxidant and Antiproliferative Activities of Twenty-Four Vitis vinifera Grapes. PLoS ONE, 9(8), e105146.

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Measuring Cell Growth Inhibition

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Drug concentrations that inhibited 50% of cell growth (GI₅₀) for adherent cell lines were determined using an SRB technique. All cell lines were treated for 24 h on day 2 and allowed to grow for an additional 3 days. Optical densities were measured at 540 nm with a Multiskan EX photometer from Thermo Electron Corporation using the Ascent Software version 2.6 (Thermo Labsystems Oy, Vantaa, Finland). Growth inhibition curves were plotted as the percentage of control cells and GI₅₀ values were determined by the GraphPad Prism 5 Software (San Diego, CA, USA) by fitting a sigmoidal curve with variable slope.

Kaliszczak M., van Hechanova E., Li Y., Alsadah H., Parzych K., Auner H.W, & Aboagye E.O. (2018). The HDAC6 inhibitor C1A modulates autophagy substrates in diverse cancer cells and induces cell death. British Journal of Cancer, 119(10), 1278-1287.

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Bacterial Growth Kinetics Monitoring

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The growth was monitored by measuring each culture’s OD_595nm periodically by use of the Multiskan Ascent Platereader with Ascent software version 2.6 (Thermo Labsystems, Thermo Fisher Scientific). Immediately after combining the bacterial liquid culture (or blank IDL medium) with the equilibrated mix of medium and treatment, 200 µL of each mixture was dispensed in quadruplicate in a 96-well flat bottom multititer plate (Greiner Bio-one). After covering with a sterile Breath-Easy® film (Sigma Aldrich), the plate was incubated at 30 °C during 48 h. Growth in each well was assessed by measuring OD_595nm every 20 min. Every 10 min the plate was shaken at speed 60 rpm during 10 seconds. Growth curves are presented as the mean OD values ± standard deviations in function of time. To reduce clutter on the graph, the standard deviation was not shown at each time point.

De Leersnyder I., De Gelder L., Van Driessche I, & Vermeir P. (2018). Influence of growth media components on the antibacterial effect of silver ions on Bacillus subtilis in a liquid growth medium. Scientific Reports, 8, 9325.

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Ferret IFN-γ Response to H7N9 Virus

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Ferret lung and spleen cells were pelleted by centrifugation at 1,500 × g_max for 5 min and washed in PBS. Cells (1.25 × 10⁴/96-well) were cultured with or without live H7N9 virus for 48 h at 37°C/5% CO₂. The Ferret IFN-γ ELISA Development Kit (ALP) (Mabtech, Stockholm, Sweden) was used to determine the quantity of IFN- γ secreted by cells ex vivo at endpoint and after restimulation with live H7N9 virus (MOI 0.1). ELISA plates were measured using a Multiskan Ascent Plate Reader with Ascent Software Version 2.6 (ThermoFisher, Waltham, MA, USA). IFN-γ-producing cells were detected using a ferret IFN-γ ELISpot assay, as per the manufacturer's instructions (Mabtech, Stockholm, Sweden).

Horman W.S., Nguyen T.H., Kedzierska K., Butler J., Shan S., Layton R., Bingham J., Payne J., Bean A.G, & Layton D.S. (2020). The Dynamics of the Ferret Immune Response During H7N9 Influenza Virus Infection. Frontiers in Immunology, 11, 559113.

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Cytokine Array for Collagen Density

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To describe a cell signaling mechanism for collagen density changes in response to high COX-2 levels and to COX-2 inhibition, a mouse cytokine ELISA plate array (Signosis, Sunnyvale, CA, USA) was used. In this quantitative chemiluminescence plate array, 23 mouse cytokines were monitored simultaneously for their expression levels in relation to collagen deposition and COX-2 inhibition with celecoxib. The cytokine signal was measured with a fluorometer (Fluoroskan, Ascent, FL) and Ascent software version 2.6 (Thermo Scientific). To compare fold-change differences, data were normalized to a blank and graphically represented by normalization to PyMT vehicle cytokine data levels.

Esbona K., Inman D., Saha S., Jeffery J., Schedin P., Wilke L, & Keely P. (2016). COX-2 modulates mammary tumor progression in response to collagen density. Breast Cancer Research : BCR, 18, 35.

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Cytotoxicity of CPT and 3-MA in NSCLC

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CPT and 3-MA were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). CPT was dissolved in dimethyl sulfoxide (DMSO) to form a stock concentration of 5 mM, and 3-MA was freshly dissolved in ddH₂O at 20 mM. Cells were treated with 0.5, 1, 2 and 5 µM CPT, and co-treated with 0.1, 0.5, 1 and 5 mM 3-MA for 24 h at at 37°C in a humidified incubator containing 5% CO₂. To determine the IC₅₀ values of CPT, 3×10³ H1299 or H460 cells/well in 96-well plates were used. After 48 h of plating, cells were treated with various doses of CPT (0.01, 0.1, 0.5, 1, 10, 50, 100 or 200 µM) for 24 h. The cells were incubated for 4 h with DMEM containing 0.5% FBS and 0.5 mg/ml MTT at 37°C in an atmosphere containing 5% CO₂ to determine the number of surviving cells. After removing the medium containing MTT and replacing with 100 µl DMSO, absorbance was recorded at 570 nm using a Multiskan Ascent microplate reader (Thermo Fisher Scientific, Inc.). The IC₅₀ values of each agent were determined using Ascent Software, version 2.6 (Thermo Fisher Scientific, Inc.).

Chiu Y.H., Hsu S.H., Hsu H.W., Huang K.C., Liu W., Wu C.Y., Huang W.P., chen J.Y., Chen B.H, & Chiu C.C. (2018). Human non-small cell lung cancer cells can be sensitized to camptothecin by modulating autophagy. International Journal of Oncology, 53(5), 1967-1979.

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Mincle-Ig Binding to Trehalose Glycolipids

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Trehalose glycolipids (4c and 4f) were coated on plates and incubated with mMincle-Ig or Ig alone [at a concentration of 3 μg/ml in binding buffer (1% BSA/TSM, BSA: bovine serum albumin; TSM buffer (pH7.0): 20 mM Tris-HCl, 1 mM CaCl₂, 150 mM NaCl and 2 mM MgCl₂)]. The preparation of Mincle fusion proteins was undertaken as described previously (Yamasaki et al., 2008 (link)). TDM (Sigma-Aldrich) and TDB were used as positive controls. Ligand bound protein mMincle-Ig was then detected using HRP-labelled anti-human Ig antibody via ELISA by optical density (OD) measurement at 450 nm [Multiskan JX and Ascent Software version 2.6 (Thermo Fisher Scientific)] and analyzed using Microsoft Excel (Microsoft).

Manthrirathna M.A., Dangerfield E.M., Ishizuka S., Woods A., Luong B.S., Yamasaki S., Timmer M.S, & Stocker B.L. (2022). Water-soluble trehalose glycolipids show superior Mincle binding and signaling but impaired phagocytosis and IL-1β production. Frontiers in Molecular Biosciences, 9, 1015210.

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Fluorescence-based Aptamer Characterization

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All fluorescence measurements were performed in the standard 96-well opaque microplates (Thermo Fisher Scientific, Denmark). Fluoroskan Ascent FL 2.6 equipped with an Ascent software version 2.6 (Thermo Scientific, Finland) was used to carry out the FAM’s fluorescence single-point measurements with excitation and emission wavelengths of 485 and 538 nm, respectively. The final volume per well was fixed at 200 μL.
Before each experiment, the aptamer solution was placed in a thermocycler (Mastercycler personal Eppendorf VWR, Leuven, Belgium) with the following temperature profile: heating at 90 °C for 8 min to the initial denaturation step, followed by a structure maintain step at 4 °C for 5 min, then a stabilization step at room temperature for 15 min.
A UV-visible spectrophotometer (UV-1800, Shimadzu, Japan) equipped with the TCC controller (TCC 240A) was used to measure the absorption characteristics of FAM-APT, DNA duplex, and target-induced aptamer folding. For this purpose, a Nanodrop cuvette (Traycell, Hellma) was used to record UV spectra with minimal samples volume fixed at 3 µL and a dilution factor cap of 10.
Fluorescence spectroscopy measurements for concept validation were carried out using an FP-8300 Spectrofluorometer from JASCO International Co.(Tokyo, Japan).

Ben Aissa S., Mastouri M., Catanante G., Raouafi N, & Marty J.L. (2020). Investigation of a Truncated Aptamer for Ofloxacin Detection Using a Rapid FRET-Based Apta-Assay. Antibiotics, 9(12), 860.

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Cytokine and Mediator Quantification

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Quantification of macrophage-derived chemokine (MDC or CCL22), CCL20, ET-1, and IL-10 was performed using ELISA kits from R&D Systems (DMD00, DM3A00, DET100, and D1000 B, respectively, Minneapolis, MN, USA). Quantification of TXA 2 , IL-33, and calcitonin was performed using ELISA kits from Biomatik (EKU07649, Kitchener, Ontario, Canada), abcam (ab108918, Cambridge, UK), Thermo Scientific (EHIL33, Waltham, MA, USA), and Cusabio (CSB-E05131h, Houston, TX, USA), respectively. All ELISAs were performed according to the manufacturer's instructions. The optical densities (O.D.) were read at 450 nm with a wavelength correction at 540 nm in a Multiscan Ascent V1.24 with Ascent Software version 2.6 (Thermo Scientific, Waltham, MA, USA). Data values were expressed as pg/mL deduced from the standard curve after subtracting the blanks, using a 4parameter logistic algorithm.

Vihlborg P., Lundberg O., Pettersson-Pablo P., Johansson N., Bryngelsson I.L., Stjernbrandt A, & Graff P. (2024). Blood biomarkers for occupational hand-arm vibration exposure. Toxicology and industrial health, 40(8).

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