Iron assay kit (MAK025, Sigma, USA) was used directly to detect both total and/or reduced iron concentrations in the samples after addition of acidic buffer. The released iron is reacted with a chromagen resulting in a colorimetric (593 nm) product, the intensity of which is proportional to the iron presented in the cells. After centrifugation at 16,000 × g for 10 min at 4 ℃, the supernatant was discharged. To measure total iron, 50 μL samples were supplemented with 5 μL iron reducer to reduce Fe3+ to Fe2+ and then adjusted to a final volume of 100 μL per well in a 96-well plate with assay buffer. Following a mixture on a horizontal shaker and incubation for 30 min at 25 ℃, 100 μL iron probe was respectively added to each well containing standard and test samples. Thereafter, the samples in the 96-well plate were mixed again and incubated for another 60 min at 25 ℃ in the dark. Finally, the absorbance was measured at 593 nm using a Spectra Max M3 Fluorescence Microplate Reader (Molecular Devices, USA).
Spectra max m3 fluorescence microplate reader
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax M3 Fluorescence Microplate Reader is a multi-mode microplate reader that can detect fluorescence in microplates. It is designed to measure various fluorescent assays.
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5 protocols using spectra max m3 fluorescence microplate reader
1
Quantifying Total and Reduced Iron Levels
2
Measuring ACE2 Activity in L. paracasei
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To measure ACE2 activity in L. paracasei, bacterial protein was extracted using the lysozyme treatment method described by Sieo et al.90 In brief, the cell pellet was suspended in 0.15 M Tris/HCl buffer (pH 6.8). Lysozyme (10 mg/mL) was added and incubated at 37°C for 90 min, followed by sonication and centrifugation at 8,000 rpm for 10 min at 4°C. The protein concentration in the supernatant was determined using a BCA protein assay kit (Pierce, Thermo Fisher Scientific, Rockford, IL, USA). To measure mouse tissue ACE2 activity, proteins were extracted by sonication in ACE2 assay buffer and centrifugation at 8,000 rpm for 10 min at 4°C as described before.35 (link) The ACE2 activity assay was performed using 50 μg of extracted protein or 10 μL of serum diluted in assay buffer (75 mM Tris, 1 M NaCl, 0.5 μM ZnCl2 [pH 7.5]) in black 96-well opaque plates with 50 μM ACE2-specific fluorogenic peptide substrate (R&D Systems, Minneapolis, MN) in a final volume of 100 μL per well reaction mixture. The fluorescent intensity was measured using SpectraMax M3 fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA) for every 90 s with excitation at 340 nm and emission at 400 nm at 37°C for 2 h, as described previously.35 (link) Experiments were carried out in duplicate, and results were expressed as relative fluorescent units (RFU).
3
Intracellular ROS Measurement via DCFH-DA
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2′,7′-dichlorodihydrofluorescein diacetate(DCFH-DA) staining (Sigma, USA) was used to measure intracellular ROS. Briefly, after treatment with different concentrations of USIONPs for 48 h, the cells were incubated with 20 μmol/L DCFH-DA for 30 min at 37 °C. Thereafter, the cells were washed twice with PBS and the fluorescence intensities at an emission wavelength of 535 nm and an excitation wavelength of 485 nm were detected using a Spectra Max M3 Fluorescence Microplate Reader (Molecular Devices, USA). Data were expressed as a percentage of the fluorescence intensity relative to vehicle controls.
4
Serum ACE and ACE2 Activity Assays
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Serum ACE and ACE2 activities were determined using assays based on fluorescent substrates, Abz-Phe-Arg-Lys[Dnp]-Pro-OH, M-2590 for ACE (Bachem, Torrance, CA), and Abz-Ser-Pro-3-nitro-Tyr-OH, M-2660 for ACE2 (R&D Systems, Inc., Minneapolis, MN), as published previously (13–15 (link)). Briefly, for ACE activity, the assay was performed in black 96-well plates with 10 μM fluorescent substrate in a final volume of 100 μL per well. For each well, 10 ul serum sample was loaded along with the assay buffer (75 mM Tris, 1 M NaCl, 0.5 μM ZnCl2, pH 7.5). The fluorescent intensity was measured using SpectraMax M3 fluorescence microplate reader (Molecular Devices) every 90 seconds with excitation at 320 nm and emission at 420 nm at 37°C for 2 hours.
The ACE2 activity assay was performed in black 96-well plates with 50 μM ACE2-specific fluorogenic peptide substrate (M-2660) in a final volume of 100 μL per well. For each well, 10 ul serum sample was loaded along with the assay buffer (75 mM Tris, 1 M NaCl, 0.5 μM ZnCl2, pH 7.5). The fluorescent intensity was measured using SpectraMax M3 fluorescence microplate reader (Molecular Devices, LLC, Sunnyvale, CA) for every 90 seconds with excitation at 340 nm and emission at 400 nm at 37°C for 2 hours.
All samples were run in duplicate and results are expressed as relative fluorescent units (RFU).
The ACE2 activity assay was performed in black 96-well plates with 50 μM ACE2-specific fluorogenic peptide substrate (M-2660) in a final volume of 100 μL per well. For each well, 10 ul serum sample was loaded along with the assay buffer (75 mM Tris, 1 M NaCl, 0.5 μM ZnCl2, pH 7.5). The fluorescent intensity was measured using SpectraMax M3 fluorescence microplate reader (Molecular Devices, LLC, Sunnyvale, CA) for every 90 seconds with excitation at 340 nm and emission at 400 nm at 37°C for 2 hours.
All samples were run in duplicate and results are expressed as relative fluorescent units (RFU).
5
Intracellular Lipid ROS Measurement
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C11-BODIPY(Thermo, USA),as a fluorescent lipid peroxidation reporter molecule that shifts its fluorescence from red to green, was used to measure intracellular lipid ROS according to the instruction of manufacture. Briefly, after treatment with different concentrations of USIONPs for 48 h, the cells were then incubated with 100 μmol/L C11-BODIPY for 30 min at 37 °C. The samples were washed twice with PBS and then the fluorescence intensities were detected at an emission wavelength of 510 nm and an excitation wavelength of 488 nm using a Spectra Max M3 Fluorescence Microplate Reader (Molecular Devices, USA). Data were expressed as a percentage of the fluorescence intensity relative to vehicle controls.
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