MLE-12 cells were seeded at 8×105 cells per 10 cm dish at 37°C for overnight, and 20μM atazanavir (Sigma-Aldrich, catalog #SML1796-5MG) was added two times at 24 h intervals. Simultaneously, MLE-12 cells were incubated in hypoxia (0.1%) for 96 h. Endogenous β-galactosidase activity was measured by the β-Gal Activity Assay Kit (BioVision, catalog# K821-100). Briefly, MLE-12 cells were lysed using 100 μl ice cold β-Gal assay buffer for 15 min after atazanavir and hypoxia treatments for 96 h. Supernatant was collected after centrifugation at 10,000 X g for 10 min at 4°C. About 10 μl of supernatant was used along with β-Gal substrate and fluorescence was measured using Spectramax i3 fluorometer (Molecular Devices, Inc.) in kinetic mode for 5 – 60 min at 37 oC.
Spectramax i3 fluorometer
Manufactured by Molecular Devices
The SpectraMax i3 fluorometer is a versatile lab equipment designed for fluorescence detection and analysis. It features a compact and modular design, allowing for flexibility in experimental setups. The SpectraMax i3 offers high-performance fluorescence measurement capabilities, catering to a wide range of applications in the scientific research and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Quaint ittm protein assay kit, by Thermo Fisher Scientific (2 mentions)
Atazanavir, by Merck Group (1 mentions)
β gal activity assay kit, by Abcam (1 mentions)
Rna later solution, by Roche (1 mentions)
Quaint it protein assay kit, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 3k, by Merck Group (1 mentions)
Bx50 light microscope, by Olympus (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
5 protocols using spectramax i3 fluorometer
1
Assessing Senescence in Hypoxic Lung Cells
2
Isolation and characterization of Eustrongylides nipponicum from Cyprinus carpio
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Adult E. nipponicum were collected from C. carpio caught in the littoral zone of the Mušov lowland reservoir (48°53′12″N, 16°34′37″E; Czech Republic). Soluble worm extract and excretory-secretory products were prepared as described previously5 (link), whereby the protein concentration was determined using the Quaint-iTTM Protein Assay Kit (Life Technologies) and a SpectraMax i3 fluorometer (Molecular Devices). Samples of the E. nipponicum tissue for isolating the RNA were immersed in RNAlater® Solution (Roche). All samples were stored in −80 °C.
Ethics statement: All procedures performed in studies involving animals were carried out in accordance with European Directive 2010/63/EU and Czech laws 246/1992 and 359/2012 which regulate research involving animals. All experiments were performed with the legal consent of the Animal Care and Use Committee of Masaryk University and of the Research and Development Section of the Ministry of Education, Youth, and Sports of the Czech Republic.
Ethics statement: All procedures performed in studies involving animals were carried out in accordance with European Directive 2010/63/EU and Czech laws 246/1992 and 359/2012 which regulate research involving animals. All experiments were performed with the legal consent of the Animal Care and Use Committee of Masaryk University and of the Research and Development Section of the Ministry of Education, Youth, and Sports of the Czech Republic.
3
Extraction and Purification of Excretory-Secretory Proteins from Adult Worms
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ESP were collected from 100 adult worms. Worms were gently washed in sterile tap water and incubated in 10 mM PBS, pH 7.2, for three hours at room temperature in Eppendorf tubes. ESP were purified and concentrated 20 times by centrifugation using an Amicon Ultra 3 kDa column (Merck Millipore) to a final volume of 5 ml. Protein concentration (0.01 μg · μl− 1) was determined using Quaint-iT Protein Assay Kit (Life Technologies) and SpectraMax i3 fluorometer (Molecular Devices). The ESP sample was stored at − 80 °C until used.
4
Isolation and Characterization of Eudiplozoon nipponicum
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Adults of E. nipponicum were collected from freshly sacrificed specimens of Cyprinus carpio caught in the Mušov lowland reservoir, Czech Republic (48°53′12″N, 16°34′37″E). Isolation of the individual worms from the gills was performed under a TH4-200 stereomicroscope (Olympus). Living worms were taxonomically identified under a BX50 light microscope (Olympus) equipped with a differential interference contrast (Nomarski DIC), and species identity was confirmed by sequencing [62 (link)].
For the preparation of excretory-secretory products (ESPs), worms (app. 100 individuals) were gently washed in 10 mM PBS pH 7.2 and incubated in fresh buffer of the same composition for 3 h at room temperature (RT). Samples were concentrated on Amicon® Ultra 3K centrifugal filters (Merck Millipore). Crude worm extract (CWE) was prepared by gently washing E. nipponicum adults (five individuals) in 10 mM PBS pH 7.2 and manually homogenised in 0.1 M acetate buffer pH 5, sonicated with BioLogics 150 VT ultrasonic homogeniser (60% amplitude, three cycles of 30 s), centrifuged (16 000×g, 20 min, 4 °C), and the supernatant was collected.
Protein concentration in ESP and CWE was determined using a Quaint-iTTM Protein Assay Kit (Life Technologies) and SpectraMax i3 fluorometer (Molecular Devices). Prior to their use, samples were stored at −80 °C.
For the preparation of excretory-secretory products (ESPs), worms (app. 100 individuals) were gently washed in 10 mM PBS pH 7.2 and incubated in fresh buffer of the same composition for 3 h at room temperature (RT). Samples were concentrated on Amicon® Ultra 3K centrifugal filters (Merck Millipore). Crude worm extract (CWE) was prepared by gently washing E. nipponicum adults (five individuals) in 10 mM PBS pH 7.2 and manually homogenised in 0.1 M acetate buffer pH 5, sonicated with BioLogics 150 VT ultrasonic homogeniser (60% amplitude, three cycles of 30 s), centrifuged (16 000×g, 20 min, 4 °C), and the supernatant was collected.
Protein concentration in ESP and CWE was determined using a Quaint-iTTM Protein Assay Kit (Life Technologies) and SpectraMax i3 fluorometer (Molecular Devices). Prior to their use, samples were stored at −80 °C.
5
Intracellular Ca2+ Dynamics in Trypanosoma Epimastigotes
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Epimastigotes (2.5 x 106 cell/mL) were incubated at different times for short-term (0, 1, 3 and 6 h) and long-term (four days) measurements of intracellular Ca2+ levels in the presence or absence (control) of 1.1 μM or 2.2 μM A6 (approximately 0.5 x IC50 and 1 x IC50). Then the parasites (1.0x107 cells) were washed with Phosphate Buffered Saline (PBS) and incubated with 5 μM Fluo-4 AM (Invitrogen) for one hour at 28° C, washed twice with HEPES glucose (50 mM HEPES, 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM glucose and 2 mM CaCl2, pH 7.4), resuspended in the same buffer and aliquoted into 96-well plates (2.5x107 per well) [30 (link)]. Readings were performed on a Spectra Max I3 fluorometer, (Molecular Devices) at λexc 490 nm and λem 518 nm. Each assay was developed in triplicate and the results correspond to the mean of three independent experiments.
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