Extracellular ROS production was determined by measuring ROS released into the medium using an Acridan Lumigen PS-3 assay 39 (link). RAW 264.7 cells (5 × 103 cells/well) were seeded in clear-bottom, 96-well black plates and treated with different concentrations of LPS (0, 1, 5, 10 μg/ml) for 1 hour. Thereafter, 50 μl aliquots of medium were mixed with the same volume of a 40:1 ratio of Reagent A (H2O2 in Tris buffer) and Reagent B (acridan solution in dioxane and ethanol) in an Amersham ECL Plus kit (GE Healthcare), and then incubated for 5 minutes at RT with light protection. Luminescence intensity at an emission wavelength of 430 nm was measured using a microplate reader (SpectraMax Gemini XPS; Molecular Devices). Intracellular ROS were measured using 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dye, according to the manufacturer's instructions. After incubating for 24 hours in clear-bottom, 96-well black plates, RAW 264.7 cells were incubated with 20 μM DCFDA dye for 45 minutes at 3 °C in the dark and then washed gently with medium. Thereafter, PBS or 5 μg/ml LPS, with or without each SPION (Fe, 1 mM), was added, and fluorescence intensity was measured at excitation and emission wavelengths of 485 and 535 nm, respectively, using a fluorescence microplate reader (SpectraMax Gemini XPS; Molecular Devices).
Spectramax gemini xps
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax Gemini XPS is a versatile microplate reader that measures absorbance, fluorescence, and luminescence. It features dual-monochromator optics for flexible assay development and high-performance detection.
Automatically generated - may contain errors
Lab products found in correlation
H2dcfda, by Thermo Fisher Scientific (5 mentions)
4 methylumbelliferyl β d glucopyranoside, by Merck Group (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Nicolet 6700 ft ir, by Thermo Fisher Scientific (3 mentions)
Fluor de lys developer, by Enzo Life Sciences (2 mentions)
Softmax pro, by Molecular Devices (2 mentions)
Enzyme assay kits, by BPS Biosciences (2 mentions)
Tcs spe, by Leica (2 mentions)
2 nbdg, by Thermo Fisher Scientific (2 mentions)
Softmax pro 6, by Molecular Devices (2 mentions)
Dcfh da, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Bradford assay, by Bio-Rad (1 mentions)
2 nitrophenyl β d galactopyranoside, by Merck Group (1 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
Thp 1 monocytes, by American Type Culture Collection (1 mentions)
Dulbecco s pbs dpbs, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated dextran, by Merck Group (1 mentions)
Fluor de lys sirt1 substrate, by Enzo Life Sciences (1 mentions)
Benchmark plus, by Bio-Rad (1 mentions)
117 protocols using spectramax gemini xps
1
Extracellular and Intracellular ROS Measurement
2
Lipid Mixing Assay for Membrane Fusion
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Lipid mixing assays were performed as previously described with minor modifications (Lee et al., 2015 (link); Lee et al., 2019 (link)). Briefly, labeled donor proteoliposomes and unlabeled acceptor proteoliposomes were mixed at a molar ratio of 1:5, transferred to a black polystyrene 384-well plate, and incubated at 30°C for 10 min. The reaction was initiated by adding 1 mM GTP and 1 mM Mg2+. NBD fluorescence was measured every minute using a SpectraMax Gemini XPS plate reader (Molecular Devices). After 40 min, β-octygludoside (final concentration 90 mM) was added to determine the total NBD fluorescence in the sample. Fusion was expressed as the percentage of total fluorescence.
3
Peroxynitrite Scavenging Activity Assay
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Peroxynitrite (ONOO−) scavenging activity was carried ou with slight modifications [59 (link)]. Shortly, 10 µL of sample solutions/positive control (penicillamine) were mixed with rhodamine buffer (pH 7.4) containing 50 mM sodium phosphate dibasic, 50 mM sodium phosphate monobasic, 90 mM sodium chloride, 5 mM potassium chloride, and 100 μM DTPA. The final DHR 123 concentration was 5 µM. The fluorescent intensities were measured after 5 min with/without authentic ONOO− using a fluorescence microplate reader (Spectramax Gemini XPS, Molecular Devices) at wavelenghts of 485 and 530 nm of excitation and emission, respectively.
4
Measuring GCase Enzymatic Activity
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GCase enzymatic activity was measured using the fluorogenic substrate 4-methylumbelliferyl-β-d-glucopyranoside (Sigma-Aldrich, St. Louis, MO, USA). The total amount of protein in cell lysates was determined using the Bradford assay (BioRad, Hercules, CA, USA), following manufacturer’s instructions. A total of 10 µL containing 10 µg of protein were incubated with 10 µL of substrate 5 mM in acetate buffer 0.1 M pH 4.2 at 37 °C for 3 h. The reaction was stopped with carbonate buffer 0.5 M pH 10.7, and the fluorescent product was quantified using a fluorimeter (SPECTRAmax Gemini XPS, Molecular Devices, San Jose, CA, USA) at an excitation wavelength of 365 nm and emission of 495 nm.
5
Quantification of Bacterial β-Galactosidase
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Expression of the lacZ reporter gene was performed using the fluorogenic substrate 4‐methylumbelliferyl β‐D‐galactoside (Melford Cat No. M1095) at a final concentration of 0.125 mg ml−1, as described previously (Ramsay, 2013 ). Samples were measured in a SpectraMax Gemini XPS fluorescence microplate reader (Molecular Devices) using the following settings: excitation 360 nm, emission 450 nm, cut‐off 435 nm, reading every 30 s for 20 min at 37 °C. β‐Galactosidase activity was expressed as relative fluorescent units s−1 and normalize to the OD600 nm of the corresponding sample. Alternatively, β‐galactosidase activity was measured as described previously (Miller, 1972) using 2‐Nitrophenyl β‐D‐galactopyranoside (ONPG; Sigma–Aldrich Cat No. N1127) as substrate. All the transcriptional fusion assays were carried out using S. plymuthica A153 LacZ (control) or derived mutants.
6
Enzymatic Assays for Uloborus sp. Midgut
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The protein content from Uloborus sp midgut samples was estimated by the bicinchoninic acid method68 (link) using albumin from the chicken egg as standard protein. Enzymatic assays were performed at 30 °C using a series of chromogenic and fluorescent substrates. Product reaction was measured using a spectrophotometer (SpectraMax M2—Molecular Devices) and spectrofluorometer (SpectraMax Gemini XPS—Molecular Devices), through at least four different intervals of time to ensure enzyme initial velocities. Blanks of the enzyme (enzyme plus buffer at the same assay condition) and the substrate (substrate plus enzyme solvent) were used as negative controls. Enzymes, buffers, substrates, and their final concentrations at the well plate are listed below (Table 1 ). At least, 5 different biological samples were used to each enzyme assay.
7
Monocyte-Endothelial Cell Adhesion Assay
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Monocyte-endothelial cell adhesion was assessed using CytoSelect Leukocyte-endothelium adhesion kit (Cat. No. CBA-210, Cell Biolabs, Inc., San Diego, CA, United States) following the assay protocol provided by the supplier. Briefly, 2 × 105 HRMECs were seeded on 0.2% (v/v) gelatin-coated 24-well plates (Abu El-Asrar et al., 2021a (link)). After reaching a confluent monolayer and overnight starvation, cells were stimulated with 25 ng/ml recombinant human TNF-α or 50 ng/ml recombinant human VEGF for 24 h with or without a 1-h pretreatment with 100 ng/ml TIMP-3. To investigate the capacity of TIMP-3 to inhibit the basal binding of THP-1 monocytes (American Type Culture Collection, Manassas, VA, United States) to HRMECs, overnight starved THP-1 monocytes were treated with or without 100 ng/ml recombinant human TIMP-3 for 24 h.
Next, 5 × 105 fluorescent-LeukoTracker labeled monocytic THP-1 cells were added to the HRMEC monolayer for 60 min. After washing, the remaining adherent THP-1 cells were lysed and fluorescence was measured using a SpectraMax Gemini-XPS (Molecular Devices, CA, United States) with excitation and emission wavelengths of 485 and 538 nm, respectively.
Next, 5 × 105 fluorescent-LeukoTracker labeled monocytic THP-1 cells were added to the HRMEC monolayer for 60 min. After washing, the remaining adherent THP-1 cells were lysed and fluorescence was measured using a SpectraMax Gemini-XPS (Molecular Devices, CA, United States) with excitation and emission wavelengths of 485 and 538 nm, respectively.
8
Measuring Cellular Oxidative Stress in Cybrids
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ROS levels were measured in our cybrids (n = 4 for all groups) using a variation of the methods we have previously described37 . Experiments were performed in sextuplicate. Briefly, cybrids were plated in 100 μL of standard culture media at a density of 10,000 cells/well in 2 separate 96-well, black well, clear-bottom plates, incubated overnight at 37 °C with 5% CO2, and subsequently incubated 48 h at 37 °C with 5% CO2 in either room-air or 2% O2. After incubation, media were replaced with 100 μL of 10 μM dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen) in Dulbecco’s PBS (DPBS; Gibco, ThermoFisher Scientific, Waltham, MA), and plates were incubated at 37 °C without additional CO2 for 30 min. Subsequently, H2DCFDA solution was removed and replaced with 100 μL DPBS. Fluorescence was measured using a SpectraMax Gemini XPS fluorescent plate reader (Molecular Devices, San Jose, CA) with excitation at 492 nm and emission at 520 nm. For each cybrid, values were normalized to the average fluorescence of samples cultured in room-air.
9
Intramitochondrial ROS Detection with DCFH-DA
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Intramitochondrial ROS production was detected using the non-fluorescent cell permeating compound, 2′7′-dichlorofluorescein diacetate (DCFH-DA). Intracellular esterases hydrolyzed DCFH-DA in dichlorofluorescein (DCFH). This non-fluorescent molecule is then oxidized to fluorescent dichlorofluorescein (DCF) by the action of cellular oxidants. Mitochondrial samples were treated with DCFH-DA (10 µM) for 30 min at 37 °C. After exposure to DCFH-DA, samples were washed with PBS solution with 0.2% Triton X-100. Fluorescence was measured in a plate reader (Spectra Max GEMINI XPS, Molecular Devices, USA) with excitation at 485 nm and emission at 520 nm. Values were expressed as fluorescence units/µg protein.
10
Quantifying Retinal Blood-Barrier Breakdown
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Blood-retinal barrier (BRB) breakdown in excised retinas was evaluated 4 days after intravitreal injection as previously described (37 (link)). Briefly, deeply anesthetized rats were intravenously injected with 50 mg/kg fluorescein isothiocyanate (FITC)-conjugated dextran (3–5 kDa, Sigma-Aldrich Corp., St. Louis, MO, USA). After 30 min, a blood sample was collected, and each rat was then perfused with PBS. The retinas were carefully excised, weighed and homogenized to extract the FITC-conjugated dextran. Fluorescence was measured using a spectra Max Gemini-XPS microplate reader (Molecular Devices, Sunnyvale, CA, USA) with excitation and emission wavelengths of 485 and 538 nm, respectively, with PBS as a blank. A correction for autofluorescence was made by subtracting the autofluorescence of retinal tissue from non-treated rats. The concentration of FITC-conjugated dextran in each retina was calculated from a standard curve of FITC-conjugated dextran in water. For normalization, the retinal FITC-conjugated dextran amount was divided by the retinal weight and by the concentration of FITC-conjugated dextran in the plasma. BRB breakdown was calculated using the following equation, with the results being expressed in μl/(g*h).
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