The skin samples were homogenised in RIPA buffer (MilliporeSigma, St. Louis, Missouri) and stored at −80°C prior to any testing. Vascular endothelial growth factor (VEGF) levels in the tissue homogenates were determined by ELISA (R&D Systems, Minneapolis, Minnesota) per manufacturer protocols. DNA quantification was performed using Hoechst 33258 dye in a 7.4 pH Tris‐EDTA buffer (MilliporeSigma) for 15 minutes at room temperature. Samples were run in duplicate. The fluorescence was determined using a SpectraMax fluorometer (Molecular Devices, San Jose, California) at an excitation of 365 and an emission of 458 nm. Calf thymus DNA standards (MilliporeSigma) were used for the standard curve. The DNA quantities in the tissue were normalised to protein content, which was determined by Pierce™ BCA assay (ThermoFisher, Rockford, Illinois) per manufacturer's guidelines. Duplicates of all samples were run in the assays, and the two data points were averaged together.
Spectramax fluorometer
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax fluorometer is a versatile laboratory instrument designed for quantitative fluorescence measurements. It provides accurate and reliable detection of fluorescent signals across a wide range of applications. The core function of the SpectraMax fluorometer is to measure the intensity of fluorescent light emitted by samples, enabling researchers to analyze and quantify various biomolecules and cellular processes.
Automatically generated - may contain errors
7 protocols using spectramax fluorometer
1
Quantifying VEGF and DNA in Skin Tissue
2
Fluorogenic Peptide-Based Enzyme Assays
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For each fluorogenic peptide, a reaction was performed in a 100 μL volume with buffer composed of 100 mM Hepes, 0.5% Triton X-100, 1 mM CaCl2 and 1 mM 2-mercaptoethanol pH 7.5 for furin (diluted to 10 U/mL), or PBS for trypsin (diluted to 50 ng/μL), or 25 mM MES pH 5.0 in the case of cathepsin L (diluted to 1 μg/mL), with the peptide diluted to 50 μM. Reactions were performed at 30 °C in triplicates, and fluorescence emission was measured every minute for 45 min using a SpectraMax fluorometer (Molecular Devices, Sunnyvale, CA, USA), with λex 330 nm and λem 390 nm wavelengths setting, enabling tracking of fluorescence intensity over time and calculation of Vmax of reactions.
3
Fluorogenic Peptide Assay for SARS-CoV-2 Proteases
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Fluorogenic peptide assays were performed as described previously with minor modifications (Jaimes et al., 2019 ). Each reaction was performed in a 100 μL volume consisting of buffer, protease, and SARS-CoV-2 S1/S2 WT (TNSPRRARSVA) or SARS-CoV-2 S1/S2 B.1.1.7 (TNSHRRARSVA) fluorogenic peptide in an opaque 96-well plate. For trypsin catalyzed reactions, 0.8 nM/well TPCK trypsin was diluted in PBS buffer. For furin catalyzed reactions, 1 U/well recombinant furin was diluted in buffer consisting of 20 mM HEPES, 0.2 mM CaCl2, and 0.2 mM β-mercaptoethanol, at pH 6.5, 7.0 or 7.5. Fluorescence emission was measured once per minute for 6Please0 continued minutes using a SpectraMax fluorometer (Molecular Devices, Inc.) at 30 °C with an excitation wavelength of 330 nm and an emission wavelength of 390 nm. Vmax was calculated by fitting the linear rise in fluorescence to the equation of a line.
4
SARS-CoV-2 Protease Fluorogenic Assay
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Fluorogenic peptide assays were performed as described previously with minor modifications (Jaimes et al., 2019 ). Each reaction was performed in a 100 μL volume consisting of buffer, protease, and SARS-CoV-2 S1/S2 WT (TNSPRRARSVA) or SARS-CoV-2 S1/S2 B.1.1.7 (TNSHRRARSVA) fluorogenic peptide in an opaque 96-well plate. For trypsin catalyzed reactions, 0.8 nM/well TPCK trypsin was diluted in PBS buffer. For furin catalyzed reactions, 1 U/well recombinant furin was diluted in buffer consisting of 20 mM HEPES, 0.2 mM CaCl2, and 0.2 mM β-mercaptoethanol, at pH 6.5, 7.0 or 7.5. Fluorescence emission was measured once per minute for 60 continued minutes using a SpectraMax fluorometer (Molecular Devices, Inc.) at 30 °C with an excitation wavelength of 330 nm and an emission wavelength of 390 nm. Vmax was calculated by fitting the linear rise in fluorescence to the equation of a line.
5
Fluorogenic Peptide Assays for SARS-CoV-2 Proteases
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Fluorogenic peptide assays were performed as described previously with minor modifications (33 (link)). Each reaction was performed in a 100 μl volume consisting of buffer, protease, and SARS-CoV-2 S1/S2 Wuhan-Hu-1 (WT) (TNSPRRARSVA), SARS-CoV-2 S1/S2 B.1.1.7 (TNSHRRARSVA) or SARS-CoV-2 S1/S2 A.23.1 (TNSRRRARSVA) fluorogenic peptide in an opaque 96-well plate. For trypsin catalyzed reactions, 0.8 nM/well TPCK trypsin was diluted in PBS buffer. For furin catalyzed reactions, 1 U/well recombinant furin was diluted in a buffer consisting of 20 mM HEPES, 0.2 mM CaCl2, and 0.2 mM β-mercaptoethanol, at pH 7.5. Fluorescence emission was measured once per minute for 60 continuous minutes using a SpectraMax fluorometer (Molecular Devices) at 30°C with an excitation wavelength of 330 nm and an emission wavelength of 390 nm. Vmax was calculated by fitting the linear rise in fluorescence to the equation of a line.
6
Cell Migration Assay: SC-1 and TGFβ
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A549 or HepG2 cells were subjected to treatment with 20 μM SC-1 in the presence or absence of TGFβ for 24 h. A total of 2 × 104 cells were suspended in serum-free medium and seeded into the inserts of transwell chambers (Greiner, Kremsmünster, Austria). The lower chambers were filled with medium containing 10% FBS, and the cells were incubated at 37 °C. After treatment, the migrated cells were fixed using methanol and stained with DAPI. Images of the migrated cells were captured at ×200 magnification using a Zeiss Axio Observer inverted microscope (Zeiss, Oberkochen, Germany). The DAPI intensity of the migrated cells was analyzed using a Molecular Devices Spectramax fluorometer (Molecular Devices, San Jose, CA, USA).
7
Fluorogenic Peptide Cleavage Assay for Protease Activity
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Fluorogenic peptide cleavage assays were performed as described previously (23 (link)), with minor modifications as described below. Reactions were carried out in a 96 well plate with a 100 μl volume consisting of cleavage buffer, protease, and fluorogenic peptide. Peptides were labeled with an MCA/DNP fluorescence resonance energy transfer (FRET) pair. Peptide sequences are listed in Table 1 . Trypsin catalyzed reactions acted as a positive control, where 0.8 nM/well TPCK trypsin was diluted in PBS buffer. For furin catalyzed reactions, 1 U/well recombinant furin was diluted in a buffer consisting of 20 mM HEPES, 0.2 mM CaCl2, and 0.2 mM β-mercaptoethanol, at pH 7.5. A negative control consisted of peptide suspended in buffer for furin cleavage with no protease added (not shown). Fluorescence emission was measured once per minute for 60 continuous minutes using a SpectraMax fluorometer (Molecular Devices) at 30°C with an excitation wavelength of 330 nm and an emission wavelength of 390 nm. Vmax was calculated by fitting the linear rise in fluorescence to the equation of the line.
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