The quantification of superoxide anion release was obtained following a standard protocol based on the reduction in cytochrome C [38 (link)], and the absorbance in culture supernatants was measured at 550 nm using the spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland). Specifically, 100 μL of cytochrome C (Merck, Milan, Italy) was added to all the wells, while 100 μL of superoxide dismutase (Merck, Milan, Italy) and 100 μL of cytochrome C were added to empty wells and the plate was then incubated for 30 min. After that, 100 μL was taken from each well and the absorbance was measured with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) at 550 nm. The O2 rate was expressed as the mean ± SD (%) of nanomoles per reduced cytochrome C per microgram of protein compared to the control (0 line).
Infinite 200 pro mplex
Manufactured by Tecan
Sourced in Switzerland, Japan, Italy
The Tecan Infinite 200 Pro MPlex is a multimode microplate reader designed for versatile and high-performance detection of a wide range of assays. It is capable of performing absorbance, fluorescence, and luminescence measurements.
Automatically generated - may contain errors
Lab products found in correlation
In vitro toxicology assay kit, by Merck Group (6 mentions)
Instantone elisa, by Thermo Fisher Scientific (4 mentions)
Nrg1 rat elisa kit, by FineTest (4 mentions)
Cayman s superoxide dismutase assay kit, by Cayman Chemical (3 mentions)
Ocln elisa kit, by MyBioSource (3 mentions)
Tnf α elisa kit, by Merck Group (3 mentions)
Rat elisa kit, by MyBioSource (3 mentions)
Tjp1 elisa kit, by MyBioSource (3 mentions)
Phosphate buffered saline (pbs), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
E1501, by Promega (2 mentions)
Padvantage, by Promega (2 mentions)
Pbs 1, by Merck Group (2 mentions)
Tbars assay kit, by Cayman Chemical (2 mentions)
Superoxide dismutase, by Merck Group (2 mentions)
Cytochrome c, by Tecan (2 mentions)
Cytochrome c, by Merck Group (2 mentions)
24 well plate, by Corning (2 mentions)
Rat oestrogen receptor beta erb elisa kit, by Cloud-Clone (2 mentions)
120 protocols using infinite 200 pro mplex
1
Quantification of Superoxide Anion Release
2
Quantification of Superoxide Anion Release
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The quantification of superoxide anion release was obtained following a standard protocol based on the reduction in cytochrome C [51 (link)], and the absorbance in culture supernatants was measured at 550 nm using the spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland). Specifically, 100 μL of cytochrome C (Merck Life Science, Rome, Italy) was added to all the wells, while 100 μL of superoxide dismutase (Merck Life Science, Rome, Italy) and 100 μL of cytochrome C were added to empty wells and the plate was then incubated for 30 min. After that, 100 μL was taken from each well and the absorbance was measured with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) at 550 nm. The O2 rate was expressed as the mean ± SD (%) of nanomoles per reduced cytochrome C per microgram of protein compared to the control (0 line).
3
Evaluating 3T3-L1 Cell Viability
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The 3T3-L1 fibroblast cell line was obtained from the ATCC, Manassas, Virginia, USA (CL-173). The effects of the HS matrices and HS-AgNP bionanomaterials on the viability of the 3T3-L1 line was assessed using the neutral red test, as described previously [46 (link)]. Briefly, 3T3-L1 cells were cultured under standard conditions (5% CO2 atmosphere, DMEM/F-12 medium (GibcoTM, Billings, MT, USA) + 10% FBS (GibcoTM, Billings, MT, USA). Aqueous solutions of the tested HS and HS-AgNP samples were added in the concentration range of 7.8–1000 μg/mL. Cell plates were placed in a CO2 incubator for 24 h. After washing with 1 × PBS, 40 μg/mL of neutral red (Sigma Aldrich, USA) working solution was added to the wells for 2 h at 37 °C. After triple washing, the dye was extracted using neutral red destain solution (50% ethanol 96%, 49% deionized water, 1% glacial acetic acid (Sigma Aldrich, USA). The optical density was measured at 540 nm and a reference wavelength of 650 nm using a Tecan Infinite 200 pro mplex multifunctional plate reader (Tecan Group Ltd., Mannedorf, Switzerland).
4
ROS Levels in H69 Cells with SFN
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The intracellular levels of ROS were detected using a fluorescence plate reader (Infinite 200Pro MPlex; Tecan Group, Ltd.), using 6-carboxy-2′,7′-dichlorofluorescein diacetate dye (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The H69 cells (3×105 cells), treated with 20 µM SFN, and untreated control cells were cultured at 37°C for 4, 8, 12, 16 or 24 h before subsequent analysis.
5
Synthesis and Characterization of SiPc(COOH)4
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All solvents, chemicals, buffer salts, and nutrient broths were purchased from Fisher Scientific, TCI, Sigma-Aldrich, Acros Organics, or Alfa Aesar and used as received. Ultrapure deionized water used for all media and buffers was provided by Thermo Scientific Barnstead Gen-Pure UV/UF Systems. Steady-state absorption and emission spectra were recorded in an Infinite 200 Pro M Plex multi-mode microplate reader (Tecan Group Ltd., Zürich, Switzerland). SiPc(COOH) 4 was synthesized using previously published methods [7, 34] .
6
Characterization of SARS-CoV-2 Spike Mutants
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SARS-CoV-2 spike mutants (D614G, D614G+W152C, D614G+L452R, and D614G+N501Y) were cloned using standard site-directed mutagenesis and PCR. Pseudoviruses typed with these spike mutants were generated as previously described with modifications (Crawford et al., 2020 ). Briefly, 293T cells were transfected with plasmid DNA (per 6-well plate: 340 ng of spike mutants, 1 μg CMV-Gag-Pol (pCMV-dΔR8.91), 125 ng pAdvantage (Promega), 1 μg Luciferase reporter) for 48 h. Supernatant containing pseudovirus particles was collected, filtered (0.45 μm), and stored in aliquots at −80°C. Pseudoviruses were quantified with a p24 assay (Takara #632200), and normalized based on titer prior to infection for entry assays.
Human airway organoids (HAO) stably expressing ACE2 (HAO-ACE2) or 293T cells stably expressing ACE2 and TMPRSS2 (293T-ACE2-TMPRSS2) were infected with an equivalent amount of the indicated pseudoviruses in the presence of 5-10 ug/ml of polybrene for 72h. Pseudovirus entry was assayed using a luciferase assay (Promega #E1501) and luminescence was measured in a plate reader (TECAN, Infinite 200 Pro M Plex). Two independent experiments were run for the 293T pseudovirus assays (2 biological replicates), with 3 technical replicates run per experiment. The HAO pseudovirus assays were run as a single experiment with 3 technical replicates.
Human airway organoids (HAO) stably expressing ACE2 (HAO-ACE2) or 293T cells stably expressing ACE2 and TMPRSS2 (293T-ACE2-TMPRSS2) were infected with an equivalent amount of the indicated pseudoviruses in the presence of 5-10 ug/ml of polybrene for 72h. Pseudovirus entry was assayed using a luciferase assay (Promega #E1501) and luminescence was measured in a plate reader (TECAN, Infinite 200 Pro M Plex). Two independent experiments were run for the 293T pseudovirus assays (2 biological replicates), with 3 technical replicates run per experiment. The HAO pseudovirus assays were run as a single experiment with 3 technical replicates.
7
Bax Activity Quantification in Chondrocytes
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BAX activity was determined in chondrocyte lysates using an ELISA kit (Human Bax ELISA Kit, MyBiosource, San Diego, CA, USA) according to the manufacturer’s instructions [89 (link)]. The absorbance of the samples was measured at 450 nm by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) and the results were compared to the standard curve (range from 0 to 2000 pg/mL) and expressed as means ± SD (%) normalized to control value (0 line).
8
Quantification of Occludin in CaCo-2 Cells
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The Human Occludin ELISA kit (OCLN kit, MyBiosource, San Diego, CA, USA) analyzed the occludin presence in CaCo-2 cell lysates, according to the manufacturer’s instruction [66 (link)]. Briefly, CaCo-2 cells were lysed with cold Phosphate-Buffered Saline (PBS, Merck Life Science, Rome, Italy) 1×, centrifuged at 1500× g for 10 min at 4 °C, and 100 μL of each sample was transferred to the strip well before the incubation at 37 °C for 90 min. The supernatants were removed, and the strips were incubated with 100 μL of Detection Solution A for 45 min at 37 °C; then, the strips were washed with Wash Solution and incubated with 100 μL of Detection Solution B for an additional 45 min. At the end of this time, 90 μL of Substrate Solution was added followed by an incubation for 20 min at 37 °C in the dark, and then 50 μL of Stop Solution was used to block the enzymatic reaction. The plate was analyzed by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) at 450 nm. The concentration is expressed as pg/mL compared to a standard curve (range from 0 to 1500 pg/mL) and the results are expressed as percentage (%) versus control (0 line).
9
Quantification of Hyaluronic Acid in Material Samples
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The assay performed to quantify the concentration of HA on material samples was the same reported in the literature [63 (link)]. Briefly, 1 mg of raw sample was dissolved in 1 mL of deionized water, and 200 µL of resuspended samples were displaced in new Eppendorf, diluted with 1.2 mL of sulfuric acid (Merck Life Science, Rome, Italy) with 0.0125 M tetraborate (Merck Life Science, Rome, Italy), shaken for 20 s and then boiled at 100 °C for 5 min. Once the samples were allowed to cool on ice, 20 µL of 0.15% hydroxydiphenyl (Merck Life Science, Rome, Italy) (dissolved in 0.5% NaOH, Merck Life Science, Rome, Italy) was added and stirred; 100 µL of each sample was placed in a 96 multi-well plate and the absorbance was measured at 340 nm by a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland). The data obtained were compared to a calibration curve generated using glucuronic acid (0, 0.25, 0.5, 1, 1.5, 2 mg/mL Merck Life Science, Rome, Italy) [64 (link)] and the results were expressed as mean (%w/w) ± SD compared to control (0 line).
10
Crystal Violet Staining for Cell Viability
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At the end of stimulation time, the cells were fixed with 1% glutaraldehyde (Merck Life Science, Rome, Italy) for 15 min at room temperature, washed, and stained with 100 µL 0.1% aqueous crystal violet (Merck Life Science, Rome, Italy) for 20 min at room temperature and solubilized with 100 µL 10% acetic acid before reading the absorbance at 595 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland). The estimated number was determined by comparing data to the control cells normalized to T0 (measurement at the beginning of the stimulation) [83 (link)]. The results were expressed as percentage (%) versus control (0 line).
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