Bone marrow derived neutrophils or splenocytes were plated on Cell-Tak coated Seahorse culture plates (300.000 cells/well) in Seahorse XF RPMI medium (pH 7.4). XF analysis was performed at 37°C with no CO2 using the XF-96e analyzer (Agilent) as per manufacturer’s instructions. Mitochondrial stress test assay was performed using Seahorse XF RPMI medium (pH 7.4) supplemented with 25 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine. Cells were treated serially with oligomycin (5 μM), FCCP (1 μM), and rotenone (100 nM) and antimycin A (10 uM), and oxygen consumption rates (OCR) were measured over time. The cell numbers at assay completion were normalized to DNA content using CyQuant dye (Thermofischer). Wave, Excel, and Graph Pad Prism software were used to analyze the data. Mann-Whitney U test was performed to calculate significance.
Cyquant dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CyQuant dye is a fluorescent nucleic acid stain used for the detection and quantification of cells. It binds to DNA and emits fluorescence upon excitation, allowing for the measurement of cell numbers in a sample. The CyQuant dye is a core laboratory tool for various applications, including cell proliferation assays, cell viability studies, and high-throughput screening.
Automatically generated - may contain errors
Lab products found in correlation
Xfe96 extracellular flux analyzer, by Agilent Technologies (2 mentions)
Xf96e analyzer, by Agilent Technologies (1 mentions)
Cba 105, by Cell Biolabs (1 mentions)
Wave software, by Agilent Technologies (1 mentions)
Mito stress test assay, by Agilent Technologies (1 mentions)
Collagen 1, by Merck Group (1 mentions)
Optipro, by Thermo Fisher Scientific (1 mentions)
Laminin 111, by Merck Group (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Euroclone (1 mentions)
8 protocols using cyquant dye
1
Seahorse XF Analysis of Neutrophil and Splenocyte Metabolism
2
Myogenic Index Quantification Methods
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Myogenic index methods were adapted from Kamli et al. [47 (link)]. Cells were seeded at 25,000 cell/well in 12-well plates and allowed to proliferated for 4 days prior to differentiation (this seeding volume yields approximately 30–50% confluency after 24 h). Cells used for differentiation measures were generated after 4 passages (P4). Cells were fixed in 4% formaldehyde for 15 min at room temperature and stained red with eosin, while nuclei were stained green with CyQUANT Dye (Thermo Fisher Scientific, Rockford, IL, USA, Cat # C35007). Cells were imaged at 10× magnification. Each cell line was seeded in three wells per plate with one image taken per well; values were taken from the average of the three images. Myogenic index was calculated as the percentage of nuclei within multinucleated fibers compared to total observed nuclei, as described by Kamli et al. [47 (link)]. Myogenic index is reported as both simple fusion (proportion of nuclei in fibers with 2 or more nuclei per fiber divided by total nuclei) and robust fusion (proportion of nuclei in fibers with 10 or more nuclei per fiber divided by total nuclei). With simple nucleation being an indication that the fusion process has begun and robust nucleation indicating that more complete myotubes have formed. Myogenic index scores for simple and robust nucleation are reported as a percent ± SEM.
3
Monocyte Chemotaxis Assessment in suPAR Transgenic Mice
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The spleens of C57BL/6J WT mice and suPARTg mice were mechanically disrupted through a 70 μm cell strainer, and splenic monocytes were isolated using The Mouse Monocyte Negative Selection Kit (19861; STEMCELL). The chemotaxis ability of isolated monocytes was assessed by cell migration assay (CBA-105; Cell BioLabs) according to the product manual. Briefly, the monocyte suspension was added to the upper membrane chamber. The bottom tray contained chemoattractant solution and RPMI media with or without 1000 ng/ml CCL2 (PHC1011; Life Technologies Corp.). After 4 and 8 hours, both the cells adherent to the membrane and cells in the bottom tray were collected and stained by CyQUANT dye (C7026, Thermo Fisher Scientific). Fluorescence measurement was performed with a 485/538 nm filter set and a 530 nm cutoff.
4
Seahorse XF Metabolic Profiling of PyMT Cells
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PyMT cells were seeded into Seahorse XFe-96 sensor plates (Agilent Technologies, Santa Clara, CA, USA) to produce near-confluence within 18 h. The next day, the plate was transferred to the Department of Pediatrics Bioenergetics Core for profiling on the XFe96 Extracellular Flux Analyzer. Cells were switched to an XF base medium supplemented with L-glutamine (2 mM) for the XF Cell Glyco Stress test or with glucose (10 mM), L-glutamine (2 mM), and sodium pyruvate (1 mM) for the XF Cell Mito Stress test. Cells were equilibrated for 1 h prior to analysis of the extracellular acidification rate (ECAR) or the oxygen consumption rate (OCR). After each run, the cell number was quantified using CyQUANT dye (Thermo Scientific, Waltham, MA, USA). Metabolic rates were calculated using the Seahorse XF report generator, and data were imported into Prism 9.0 for analysis.
5
Cell Quantification by DNA Content
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Quantification of cell numbers was carried out by measuring DNA content using CyQuant dye (Thermo Fisher Scientific; C7026). Fluorescence was measured with a CLARIOstar microplate reader at 485Ex/535Em. The standard curve was made with B16 and MNT-1 cells from 1000 to 32,000 cells per 100 μl sample.
6
Oxygen Consumption and Extracellular Acidification Rates
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Oxygen consumption and extracellular acidification rates were measured using an XFe96 Extracellular Flux Analyzer (Seahorse Bioscience) using the manufacturer’s established protocols. In brief, cells were plated overnight in Seahorse 96-well plates at 1 × 104 per well in 80 μL of complete media. Cells were washed three times in non-buffered DMEM containing 25 mM glucose and 2 mM glutamine and incubated in this medium in a CO2-free incubator at 37 °C for 2 h to allow for temperature and pH equilibration before loading into the XFe96 apparatus. In addition to basal measurements, the Mito Stress Test assay (Seahorse Bioscience, 103015-100) was performed according to manufacturer’s instructions. XFe assays consisted of sequential mix (3 min), pause (3 min), and measurement (5 min) cycles, allowing for determination of OCR/ECAR every 10 min. At the end of the assay media was removed from the plate which was then frozen at −80 °C for 24 h. CyQuant dye (Invitrogen, C7026) was then used according to the manufacturer’s instructions to generate DNA content values for data normalization. Data were analyzed using Wave software (Seahorse Bioscience).
7
Quantifying Fibroblast Adhesion and Migration
8
Curcumin Modulates Cadmium Toxicity
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HL-60 cells (EACC) [15] (link) were cultured in RPMI medium supplemented with 10% FBS, 1% Lglutamine and 1% penicillin/streptomycin (Euroclone, Milan, Italy) at 37 °C in a humidified atmosphere containing 5% CO2. Cell differentiation was obtained after one week supplementation with 100 nM vitamin D (VD) analogue EB1082 (Sigma/Aldrich, Milan) [14] (link). Differentiated HL-60 cells were preincbated overnight. with 1 μM Cur (Sigma/Aldrich) or 0.1% DMSO (CTRL). Day after, medium was changed and cells were incubated with 15 μM Cd Nitrate (Cd(NO3)2, Sigma/Aldrich) for additional 24 h. Cellular viability was assessed with CyQuant dye (Invitrogen, TermoFischer, Milan, Italy) as described [16] (link). After cell staining, microphotographs were captured by fluorescence inverted microscope (Axiovert Zeiss) at 400× magnification using FITC filter.
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