Cells (1×104) were seeded onto a 96-well-plate (Corning, black plate, clear bottom) in normoxia or hypoxia with appropriate phenol-red free medium. For a positive control, cells were treated with 400 μM H2O2 for 1.5h before the measurement. Treated and non-treated cells were washed with PBS and incubated with 5 μM 2’,7’dichlorofluorescein diacetate (DCF-DA-Sigma-Aldrich) for 30 min at 37°C in the dark. DCF was then detected with an Infinite F200 PRO microplate reader (TECAN Trading AG, Switzerland) using excitation and emission wavelengths of 485 and 525 nm, respectively. Four independent experiments were performed and protein normalization was made after each experiment.
Black plate
Manufactured by Corning
Sourced in United States
The Black plate is a type of laboratory equipment used in various scientific and research applications. It is a flat, rectangular surface made of a durable material, typically a dark color such as black. The primary function of the Black plate is to provide a consistent and neutral background for various experimental setups and observations, allowing for accurate visualization and analysis of samples or data.
Automatically generated - may contain errors
Lab products found in correlation
Infinite f200 pro, by Tecan (1 mentions)
Xenogen ivis, by PerkinElmer (1 mentions)
Luciferin, by Biosynth (1 mentions)
Superblock reagent, by Thermo Fisher Scientific (1 mentions)
Pierce maleic anhydride activated plates, by Thermo Fisher Scientific (1 mentions)
Ace2 activity assay kit fluorometric, by Abcam (1 mentions)
4 protocols using black plate
1
Quantifying Intracellular Oxidative Stress
2
Luciferase Assay for Promastigote Cells
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Logarithmic growth phase promastigotes (106) were suspended in 200 μL media containing 30 μg/ mL of luciferin (Biosynth AG, Staad, Switzerland) and added to a 96-well plate (Black plate, Corning Incorporated, NY, USA). The plate was imaged after 10 min using a Xenogen IVIS photoimager (Caliper LifeSciences), and luciferase activity quantitated as photons/sec (p/s).
3
FLAA Test for SARS-CoV-2 S-Protein Detection
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The FLAA test for SARS-CoV-2 S-protein detection was set in 96-well microplates as previously described [14 (link)]. Briefly, 100 pmole of 5′-amino-C6-modified C7 aptamer was immobilized on clear or black opaque Pierce™ maleic anhydride activated plates (Thermo Fisher Scientific, Waltham, MA, USA) as a capture agent. Plates were blocked using the Super-Block™ reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. Supernatant from Sal, Sw or Sw-D samples was added in TNa7 for the final 10% and 20% concentrations; a volume of 100 μL of each dilution was then poured into C7-coated plates and incubated in an orbital shaker for 1 h at room temperature. Wells were washed five times with TNa7 and then 50 pmole of the 5′-FAM-C9 aptamer were added as a detection agent. After a second incubation for 1 h at RT, plates were washed five times with TNa7, and fluorescence was either acquired immediately (direct protocol) from black opaque plates or incubated in clear plates with 150 μL of 7 M urea for 30 min at RT for C9 aptamer retrieval and posterior transference into black plates (Corning Inc., Corning, NY, USA) for further reading (indirect protocol). Finally, fluorescence in the 96-well black plates was measured at 491ex/516em nm using a BioTek® Synergy™ H4 Hybrid Multi-mode microplate reader (Thermo Fisher Scientific).
4
Quantifying ACE2 Activity in Cells and Tissues
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Activity of ACE2 in cell lines, cell lysates and tissues were measured by ACE2 activity assay kit (Fluorometric) (Abcam, UK), which utilizes the ability of an active ACE2 to cleave a synthetic MCA based peptide substrate to release a free fluorophore. The measurements were performed in black plates (Corning, USA) with a total volume of 100 μl lysates and ACE2 substrate. HUVECs were treated with 25 μM imatinib, 100 μM harpagoside and 100 μM methazolamide respectively for 16 h, then lysed and followed by measurement according to the kit. Similarly, tissues from the mice treated with imatinib or methazolamide were applied for ACE2 enzymatic activity measurement. HUVEC lysates and imatinib (1.5×10-7 M, 1.5×10-6 M), harpagoside (0.5×10-7 M, 1.5×10-6 M), or methazolamide (1.5×10-7 M, 1.5×10-6 M) were incubated respectively for 30 min at room temperature to assess the time dependency of the fluorescence signal. Fluorescence kinetics was measured by use of a multiplate reader (SpectraMax 5, USA) at an excitation wavelength of 320 nm and an emission wavelength of 420 nm. ACE2 inhibitor DX600 was used as a negative control.
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