1×106 RAW 264.7 macrophages were seeded into wells of a 6 well plate. Following overnight incubation the cells were treated with 10 mM MβCD for 1 h or with 4 μg/ml cholesterol (SyntheChol; Sigma) for 10 h. Untreated cells were used as control. The cell monolayers were washed once with PBS, dried and incubated in 1 ml isopropanol per well overnight at room temperature. The supernatant containing the extracted lipids was dried under vacuum and resuspended in reaction buffer provided with the Amplex Red Kit (Invitrogen). Cholesterol levels were determined fluorimetrically utilizing the Amplex Red Kit (Invitrogen) according to the manufacturer`s instructions and normalized to macrophage protein content. The remaining cell monolayers were solubilized in 1.5 ml/well 0.1 M NaOH and the total protein content was measured using the BCA assay. Two independent experiments were performed in duplicate.
Amplex red kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Amplex Red kit is a fluorogenic reagent used for the detection and quantification of hydrogen peroxide (H2O2) and peroxidase activity in various biological samples. The kit utilizes the Amplex Red reagent, which reacts with H2O2 in the presence of peroxidase to produce the highly fluorescent compound resorufin. This reaction can be monitored using fluorescence spectroscopy, providing a sensitive and reliable method for measuring H2O2 levels and peroxidase activity.
Automatically generated - may contain errors
Lab products found in correlation
Amplex red, by Merck Group (3 mentions)
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions)
Hts7000 plate reader, by Tecan (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Unopsonized zymosan, by MP Biomedicals (2 mentions)
Genios plate reader, by Tecan (2 mentions)
N formylmethionine leucyl phenylalanine fmlp, by Merck Group (2 mentions)
Infinite f200 pro plate reader, by Tecan (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Polarstar optima, by BMG Labtech (2 mentions)
Bradford reagent, by Bio-Rad (2 mentions)
Glomax multi detection system, by Promega (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Bodipy ceramide, by Thermo Fisher Scientific (1 mentions)
35s cysteine methionine, by Cytiva (1 mentions)
Smase from staphylococcus aureus, by Merck Group (1 mentions)
Zymosan, by Merck Group (1 mentions)
El406 plate washer, by Agilent Technologies (1 mentions)
Lps binding protein, by R&D Systems (1 mentions)
70 protocols using amplex red kit
1
Cholesterol Levels in RAW 264.7 Macrophages
2
Exosome Purification from Transfected HEK293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Twenty-four hours after HEK293T cell transfection, cultures switched to fresh complete medium containing 5% exosome-deprived FCS, which was obtained after ultracentrifugation at 70,000 × g, 3 hrs at 4°C. Cell supernatants were harvested from 48 to 72 hrs after transfection and centrifuged at 500 × g for 10 mins. Clarified supernatants were therefore processed for exosome purification by differential centrifugations as previously described.17 (link) Briefly, supernatants underwent a first ultracentrifugation at 10,000 × g for 30 mins at 4°C, followed by 0.22 μm pore-size filtration. Supernatants were then ultracentrifuged at 70,000 × g for 2.5 hrs at 4°C. Pelleted vesicles were washed with phosphate-buffered saline (PBS), and ultracentrifuged again at 70,000 × g for 1 hr at 4°C. Pellets were finally resuspended in either DMEM (1:200 of the initial supernatant volume) or 0.1% Triton-X100 TNE (1:300 of the initial volume).
Exosome preparations were quantified by measuring the activity of acetylcholinesterase (AchE), i.e., a typical exosome marker,18 (link) through the Amplex Red kit (Molecular Probes) following the manufacturer’s recommendations. The AchE activity was measured as mU/mL, where 1 mU is defined as the amount of enzyme which hydrolyzes 1 pmole of acetylcholine to choline and acetate per minute at pH 8.0 at 37°C.
Exosome preparations were quantified by measuring the activity of acetylcholinesterase (AchE), i.e., a typical exosome marker,18 (link) through the Amplex Red kit (Molecular Probes) following the manufacturer’s recommendations. The AchE activity was measured as mU/mL, where 1 mU is defined as the amount of enzyme which hydrolyzes 1 pmole of acetylcholine to choline and acetate per minute at pH 8.0 at 37°C.
3
Quantifying Vesicle-Associated AchE Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The vesicle-associated AchE activity was evaluated through the Amplex Red kit (Molecular Probes) following the manufacturer’s recommendations. The AchE activity was measured as mU/mL, where 1 mU is defined as the amount of enzyme which hydrolyzes 1 pmole of acetylcholine to choline and acetate per minute at pH 8.0 at 37°C.
4
Lipid Quantification and Separation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bodipy-sphingomyelin (Bdp-SM), Bodipy-ceramide (Bdp-cer), and Amplex Red kit were obtained from Molecular Probes (Eugene, OR). The purity of the above lipids was verified by thin-layer chromatography (TLC) on silicic acid-coated plates (Merck, Darmstadt, Germany) developed with chloroform/methanol/water (65 : 25 : 4, by vol.). The concentrations of Bdp-SM and Bdp-cer in chloroform were determined spectrophotometrically using 77,000 cm−1 and 91,000 cm−1, respectively, for their molar extinction coefficients. [35S]-cysteine/methionine was obtained from Amersham Biosciences. Proanalysis grade solvents were from Merck, SMase from Staphylococcus aureus from Sigma, and other chemicals from standard sources.
5
Assessment of NADPH-Oxidase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NADPH-oxidase activity was assessed as hydrogen peroxide release determined by an Amplex Red kit (Molecular Probes). Neutrophils (0.25 × 106/ml) were stimulated with platelet-activating factor (PAF) and Formyl-Methionyl-Leucyl-Phenylalanine (fMLP; both 1 µM, added simultaneously) and 1 mg/ml serum treated Zymosan STZ, or 100 ng/ml PMA in the presence of Amplex Red (0.5 µM) and horseradish peroxidase (1 U/ml). Fluorescence was measured at 30-s intervals for 20 min with the HTS7000+ plate reader. Maximal slope of H2O2 release was assessed over a 2-min interval. Using unopsonized zymosan, the same steps and procedures were followed, but fluorescence was measured for 60 min at similar 30-s intervals. Maximal slope of H2O2 release was assessed over a 2-min interval.
6
Measuring NADPH Oxidase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NADPH oxidase activity was measured by assaying the hydrogen peroxide production by PMNs derived from blood or from iPSC-derived neutrophils, from either the HPS2 or the WT iPSC line, in response to various stimuli with the Amplex Red kit (Molecular Probes; Life Technologies), as described before (25 (link), 38 (link)). In short, PMNs or cultured neutrophils (1 × 106/ml) were stimulated in Hepes medium (132 mM NaCl, 20 mM Hepes, 6.0 mM KCl, 1.0 mM MgSO4, 1.0 mM CaCl2, 1.2 mM potassium phosphate, 5.5 mM glucose, and 0.5% [wt/vol] human serum albumin, pH 7.4) with opsonized E. coli (0.25 × 109/ml), zymosan (1 mg/ml; Sigma-Aldrich), serum-treated zymosan (1 mg/ml), or PMA (100 ng/ml; Sigma-Aldrich) in the presence of Amplex Red (0.5 μM) and horseradish peroxidase (1 U/ml). Fluorescence was measured at 30-s intervals for 30 min with the HTS7000+ plate reader (Tecan). The maximal slope of hydrogen peroxide release was assessed over a 2-min interval to determine the activity of the NADPH oxidase.
7
Quantifying Cellular ROS Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Amplex Red kit (Molecular Probes) was used to determine the amount of reactive oxygen species produced by NB4 control or reconstituted cells, as previously described (52 (link)). 2.5 × 105 cells/mL were incubated with Amplex Red (25 μM) and horseradish peroxidase (0.5 U/mL). 5 min later, cells were stimulated with 100 ng/mL phorbol 12-myrisatate 13-acetate (PMA; Sigma-Aldrich), 1 mg/mL unopsonized zymosan (MP Biomedicals), or serum-treated zymosan (STZ), for 30 min at 37°C. Every 30 s, the fluorescence was assessed with a GENios plate reader (Tecan). The maximal slope of H2O2 release was measured with a 2-min interval at an excitation wavelength of 535 nm and an emission wavelength of 595 nm.
8
Hydrogen Peroxide Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hydrogen peroxide production was determined using an Amplex Red kit (Molecular Probes), according to the manufacturer’s instructions. In the presence of peroxidase, Amplex Red reagent reacts with H2O2 (in 1:1 stoichiometry) to produce a red fluorescent product called resoruffin. The high extinction coefficient of resoruffin allows for analysis either fluorometrically or spectrophotometrically. Aliquots of medium were subsequently removed and analyzed spectrophotometrically at a wavelength of 560 nm. After H2O2 determination, samples were washed thoroughly and corrected for cell number using a CytoSelect colormetric assay kit (Cell Biolabs, Inc., San Diego, CA). Dye from the stained cells was extracted and quantified at OD 560 nm.
9
Transfection and Viral Infection Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfection with siRNAs was performed according to manufacturers’ instructions using RNAiMAX (Thermo Fisher Scientific). We performed reverse transfection by seeding 80 μl HeLa cells (2 × 103 cells/well) onto a 20 μl mix of siRNA (10 nM) and transfection reagent. Hela MZ cells were infected with VSV 19 using a BioTek™ EL406 plate washer, fixed with 4% PFA and stained with DAPI, and images were acquired with the IXM™ microscope and analysed with MetaXpress™ to quantify infected cells. The cholesterol content of liver extracts was measured enzymatically using the Amplex Red Kit (Molecular Probes). Dried lipid samples were re‐dissolved in pure methanol, sonicated for 5 min and diluted in the assay buffer for quantification. Electron microscopy after plastic embedding for HRP analysis 61 or after immunogold labelling of cryosections 62 has been described. Quantitation of LBPA labelling of sections from control (DMSO‐treated) or thioperamide‐treated cells was performed in a double‐blind fashion by counting the number of gold particles per endosomes in each endosome identified in two sets of 16 micrographs for each condition (DMSO‐ and thioperamide‐treated cells).
10
Measuring Neutrophil NADPH Oxidase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NADPH oxidase activity was measured by assaying the hydrogen peroxide production by mature neutrophils derived from blood or neutrophil progenitors derived from bone marrow in response to various stimuli with the Amplex Red kit (Molecular Probes, Life Technologies, Carlsbad, CA, USA). Cells (1 × 106/mL). In short, cells were stimulated in Hepes-buffered saline solution with fMLF (1 μM), TNFα (10 ng/mL), LPS (20 ng/mL) + LPS-binding protein (LBP) (50 ng/mL, R&D Systems, Minneapolis, MN, USA) or PMA (100 ng/mL, Sigma) in the presence of Amplex Red (0.5 μM) and horseradish peroxidase (1 U/mL). Fluorescence was measured at 30 s intervals during 4 h with the HTS7000+ plate reader (Tecan, Zurich, Switzerland). Maximal slope of hydrogen peroxide release was assessed over a 2 min interval.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!