Reticulocyte quantification was determined by percentage of CD71-positive cells, of Thiazole Orange (TO)-positive cells, and of reticulocytes. To determine the percentage of CD71-and TO-positive cells, 1 µL of RBCs (i.e., approximately 4 × 106 RBCs) were incubated for 30 min at 4°C in the dark with a saturating concentration of an antibody mixture containing anti-CD71 (APC; BD Biosciences) and anti-CD235a (FITC; Biorad), Thiazole Orange (Sigma-Aldrich) and anti-CD235a (APC; Biorad), or IgG1 isotype negative control (FITC; ThermoFisher). Labeled cells were then washed twice, suspended in 200 µL of HEPES buffer, and acquired using the LSR Fortessa Flow Cytometer (BD Bioscience). 10,000 events were recorded for each sample. The analysis was performed with FlowJo v10 software (FlowJo, Ashland, OR). Reticulocyte percentage and RBC indices, including mean cell volume (MCV), mean cell Hb (MCH), mean cell Hb concentration (MCHC), and hemoglobin concentration (HGB), were analyzed by ADVIA 2120i Hematology System (Siemens).
Thiazole orange
Manufactured by Merck Group
Sourced in United States, France, India, Germany
Thiazole orange is a fluorescent dye that is commonly used in laboratory applications. It exhibits strong fluorescence when bound to nucleic acids, making it useful for the detection and quantification of DNA and RNA. The dye can be excited with blue or green light and emits light in the yellow-orange region of the visible spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Facscalibur flow cytometer, by BD (4 mentions)
Facscalibur, by BD (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Pen strep solution 100x, by Thermo Fisher Scientific (3 mentions)
Hoechst 33258, by Dojindo Laboratories (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Hydrochloric acid (hcl), by Merck Group (2 mentions)
Cd41 apc, by Thermo Fisher Scientific (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa, by BD (2 mentions)
Hepes, by Merck Group (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Rnase, by Carl Roth (2 mentions)
Sulforaphane, by Merck Group (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
67 protocols using thiazole orange
1
Reticulocyte Quantification Protocols
2
Synthesis and Characterization of PES-TiO2 Nanocomposites
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PES was obtained as a polymer
from Sigma-Aldrich, India. Titanium butoxide (TNBT), poly(vinylpyrrolidone)
(PVP), acetic acid, hydrochloric acid (HCl), and N-methyl-2-pyrrolidone (NMP) were purchased from Merck, India. Lysine,
(3-glycidyloxypropyl) trimethoxysilane GPMS, bovine serum albumin
(BSA), thiazole orange (TO), and propidium iodide (PI) were procured
from Sigma-Aldrich, India.
from Sigma-Aldrich, India. Titanium butoxide (TNBT), poly(vinylpyrrolidone)
(PVP), acetic acid, hydrochloric acid (HCl), and N-methyl-2-pyrrolidone (NMP) were purchased from Merck, India. Lysine,
(3-glycidyloxypropyl) trimethoxysilane GPMS, bovine serum albumin
(BSA), thiazole orange (TO), and propidium iodide (PI) were procured
from Sigma-Aldrich, India.
3
Flow Cytometry of Reticulated Platelets
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Reticulated platelets were determined by flow cytometry using thiazole orange (TO, Sigma-Aldrich, St Louis, MO, USA). Briefly, platelets were incubated with TO, 10 ng/mL for 1 hour at 22 °C in the dark. Platelets were identified using PE-conjugated anti-CD41.
4
EV Staining and Isolation
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EVs were diluted in PBS (1:20) and gently mixed in the dark with 1 mg/mL thiazole orange (Sigma-Aldrich, Rehovot, Israel) in 100% ethanol (1:2000). EVs were then separated using an ultracentrifuge 50.2 Ti fixed-angle rotor at 100,000× g, 4 °C, 12 h. The pellet was suspended with cold PBS.
5
Quantifying DNA Intercalation by Thiazole Orange
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DNA intercalation was
measured by incubating 20 nM unlabeled, undamaged oligonucleotides
in NEIL1 reaction buffer (excluding bovine serum albumin) in the presence
of 100 nM Thiazole Orange (Sigma-Aldrich 390 062) as described
in ref (115 (link)).
measured by incubating 20 nM unlabeled, undamaged oligonucleotides
in NEIL1 reaction buffer (excluding bovine serum albumin) in the presence
of 100 nM Thiazole Orange (Sigma-Aldrich 390 062) as described
in ref (115 (link)).
6
Spermidine-NBD Uptake Kinetics in P. aeruginosa
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P. aeruginosa were preincubated with 4 mM spermidine-NBD or unlabeled spermidine, following the same protocol as that used for MIC assays. Following incubation, bacteria were pelleted by centrifugation and resuspended in either PBS or CF sputum. Immediately, and at 30, 60, 120, 180, and 240 minutes, 200 μL samples were removed from the cultures and analyzed for NBD fluorescence on a FACSAria II flow cytometer (BD Biosciences). Twenty thousand individual bacteria were recorded. Side-scatter and forward-scatter limits for bacterial flow cytometry were predetermined using P. aeruginosa stained with the DNA dye thiazole orange (Sigma-Aldrich).
7
Fetal Blood Cell Staining and Imaging
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Fetal blood samples were stained with Hoechst 33342 (Molecular Probes, Eugene, OR) and MitoTracker Deep Red (Invitrogen) according to manufacturer directions. Cells were then blocked and stained in 5% rat whole serum (Invitrogen) prepared in PB2 (Dulbecco PBS and 0.1% glucose [Invitrogen] and 0.3% BSA [Gemini BioProducts]) with 1:200 dilutions of PE-CD71, Biotin-Ter119 (eBioscience, San Diego, CA), and 2 μg/mL of thiazole orange (Sigma-Aldrich). After washing, cells were stained with 1:250 PE-Texas Red streptavidin (BD Biosciences, San Jose, CA). Data were collected on the ImageStreamX and analyzed using IDEAS 6.2 (Luminex, Austin, TX); debris, calibration beads, and cell aggregates were excluded by gating on size and aspect ratios, and focused cells were selected based on sharpness of the brightfield stain (gradient root mean square) as previously described.25 (link)
8
Rat Blood and Bone Marrow Cell Purification
9
Estimation of SYBR Safe Dye Concentration
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Example 30
The 1× working concentration of SYBR Safe is not disclosed by the manufacturer. However, the dye's chemical structure has been determined to be an asymmetric cyanine dye, similar to Thiazole Orange (Evenson, et al. J Org Chem 77(23), 10967(2012)). The extinction coefficient of Thiazole Orange (Sigma-Aldrich) is determined to be ˜70,000. Using this extinction coefficient and the optical density of SYBR Safe 1× solution, the concentration of the 1× was determined to be ˜1.3 μM.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
10
Rapid Viability Assessment of Salmonella
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The flow cytometer used in this study was an Apogee Model A50 (Apogee Flow Systems Ltd., Hemel Hempstead, United Kingdom). The RAPID-B TPC method described previously can separate viable from non-viable cells due to the presence of thiazole orange (TO, Sigma-Aldrich) and propidium iodide (PI, Sigma-Aldrich) (Wilkes et al., 2012 (link); Buzatu et al., 2013 (link)). To generates green fluorescence (FL1 channel) for live cells, while PI quenches TO and emits red fluorescence (FL3 channel) for dead cells. To assess the survivability of the Salmonella serotypes, serial dilutions were treated with TPC reagent and analyzed using flow cytometry. Briefly, to a volume of 667 μL of each dilution in a 2 mL microcentrifuge tube, 333 μL of TPC reagent was added. The mixture was incubated for 5 min at room temperature, under slow shaking and then analyzed on the flow cytometer. The different dilutions of each serotype were analyzed three times, and the event counts per 100 μL were recorded. The flow cytometry results were converted to cells per mL and compared to colony forming units per mL (CFU/mL) obtained from plate counts.
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