Cell viability was determined using the Alamar blue assay [85 (link)], which reflected the mitochondrial activity. Viable cells were characterized by an effective mitochondrial metabolization of the non-fluorescent dye resazurin (Sigma, Steinheim, Germany) to fluorescent resorufin (excitation: 535 nm, emission: 590 nm). Relative viability in the untreated control was set to 100%. In addition, the Neutral red assay, which measures the integrity of lysosomal membranes, was used to monitor cytotoxicity. To this end, cells were incubated with the Neutral red solution (0.01%) in an atmosphere containing 5% CO2 for 90 min before they were fixed (1% formaldehyde/1% CaCl2). Afterwards, the Neutral red that was taken up by the cells was extracted (50% ethanol/1% of acetic acid, 15 min at room temperature (RT)) and absorbance (540 nm) was measured. If not stated otherwise, data are shown as the mean ± standard deviation (SD) of two to three independent experiments each performed in quadruplicate. Approximate IC50 (~IC50, see Supplementary Table S1 ) were calculated from the corresponding dose-response curves.
Resorufin
Manufactured by Merck Group
Sourced in United States, Germany, Czechia, Sao Tome and Principe, Switzerland
Resorufin is a fluorescent dye commonly used in biochemical and cell-based assays. It is a red-colored, water-soluble compound that emits a red-orange fluorescence when excited at the appropriate wavelength. Resorufin is often used as a substrate or indicator in various enzymatic and cell viability assays, providing a quantitative measurement of specific biological activities.
Automatically generated - may contain errors
Lab products found in correlation
Resazurin, by Merck Group (15 mentions)
Nadph, by Merck Group (14 mentions)
Methoxyresorufin, by Merck Group (12 mentions)
Ethoxyresorufin, by Merck Group (12 mentions)
P nitrophenol, by Merck Group (11 mentions)
Coumarin, by Merck Group (8 mentions)
Cytochrome c, by Merck Group (8 mentions)
Glucose 6 phosphate (g6p), by Merck Group (8 mentions)
Glucose 6 phosphate dehydrogenase, by Merck Group (8 mentions)
7 ethoxyresorufin, by Merck Group (8 mentions)
1 chloro 2 4 dinitrobenzene, by Merck Group (7 mentions)
7 hydroxycoumarin, by Merck Group (6 mentions)
Erythromycin, by Merck Group (6 mentions)
Chlorzoxazone, by Merck Group (6 mentions)
Testosterone, by Merck Group (6 mentions)
Heparin, by Merck Group (5 mentions)
4 nitrocatechol, by Merck Group (5 mentions)
Benzyloxyresorufin, by Merck Group (5 mentions)
Glutathione (gsh), by Merck Group (5 mentions)
Diclofenac, by Merck Group (4 mentions)
79 protocols using resorufin
1
Cytotoxicity Evaluation Using Viability and Membrane Integrity Assays
2
Cytochrome c Enzymatic Assays in Cells
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Cytochrome c (cyt c) (horse heart), glucose-6-phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PD), nicotinamide-adenine dinucleotide phosphate (NADPH), ethoxyresorufin, methoxyresorufin, resorufin, coumarin, 7-hydroxy coumarin, 3-cyano-7-ethoxycoumarin, 3-cyano-7-hydroxycoumarin, fluorescein, trypsin, penicillin-streptomycin (10,000 units penicillin and 10 mg streptomycin per mL), Dulbecco’s Modified Eagle’s Medium-low glucose (DMEM), fetal bovine serum (FBS), phosphate buffered saline pH 7.4 (PBS) and doxorubicin were obtained from Sigma Aldrich (St. Louis, MO, USA). Nicotinamide adenine dinucleotide phosphate (NADP+) was obtained from Gerbu (Heidelberg, Germany). Bradford reagent was obtained from Bio-Rad (Hercules, CA, USA) and Quiazol from Qiagen (Hilden, Germany). Dibenzylfluorescein, sodium dithionite, dimethyl sulfoxide (DMSO), acetonitrile (ACN) and sodium hydrogencarbonate (NaHCO3) were purchased from Merck (Kenilworth, NJ, USA). Insulin was obtained from Cell Applications, Inc. (San Diego, CA, USA). All other chemicals and solvents were of the highest grade commercially available.
3
Antimicrobial Activity Assay of M. tuberculosis
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Logarithmically growing M. tuberculosis was inoculated into 7H9 medium in 96-well plates with wells containing increasing concentrations of compound. M. tuberculosis was inoculated at an ODλ600 of 0.0008, corresponding to approximately 4 × 105 CFU/ml in 200 μl per well. The plates were incubated at 37°C in 5% CO2 for 1 week, at which point 32.5 μl of a mixture containing an 8:5 ratio of 0.6 mM resazurin (Sigma) dissolved in 1× phosphate-buffered saline to 20% Tween 80 was added, and the production of fluorescent resorufin was measured after incubation at 37°C in 5% CO2 overnight. For M. tuberculosis , samples were removed from the plate and mixed with formalin to kill the M. tuberculosis bacteria before measuring the fluorescence. For M. smegmatis , the assay plate was measured directly. Fluorescence was measured on a Tecan M200 Pro plate reader with an excitation λ of 530 nm and an emission λ of 590 nm. For each assay, medium alone served as a negative control, and untreated M. tuberculosis or M. smegmatis was included as a positive control. The percent inhibition was calculated as the {[(fluorescence of the positive control − fluorescence of the negative control) − (fluorescence of the sample − fluorescence of the negative control)]/(fluorescence of the positive control − fluorescence of the negative control)} × 100.
4
Inhibition of Candida Biofilm Formation by P. nigrum Extract
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The effect of P. nigrum ethanolic extract on the biofilm formation of C. albicans strains was tested in 96-well microplates [57 (link),58 (link)]. One hundred µL of Candida cell suspensions (1 × 106 cells/mL) in RPMI-1640 with MOPS were dispensed in 96 microdilution wells with or without P. nigrum extract (8 to 2048 μg/mL). The plates were then incubated at 37 °C and allowed to adhere for 1 h. The non-adherent cells were removed, and 200 μL of fresh RPMI was added. The plates were incubated further at 37 °C for 24 h. After incubation, biofilms were washed twice with PBS, and finally, 200 µL of RPMI-1640 plus 10 µL of 700 µM resazurin (Sigma–Aldrich) was added to each well and incubated at 37 °C for 2 h. The biofilm was quantified indirectly by measuring the fluorescent water-soluble resorufin product that results when resazurin is reduced by reactions associated with respiration. Fluorescence was measured at 560 nm with emission at 590 nm in an automated plate reader (Model 550 Microplate Reader Bio-Rad, Milan, Italy). Caspofungin (0.03 to 1 μg/mL) was used as a standard antifungal drug.
5
Resorufin Staining for Amyloid-Beta Vascular Deposits
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For the specific detection of Aβ-positive vessels, resorufin staining was performed in paraffin-embedded mice brain sections following the protocol described by Han et al. [75 (link)]. Briefly, all samples were deparaffinized, washed 3 times in PBS, and permeabilized in PBS-0.2% Triton X-100 (PBST) for 30 min. Samples were then immersed in 1 mM resorufin (Sigma-Aldrich) dissolved in PBST for 5 min. After rinsing with PBS, samples were rinsed with PBS-50% ethanol for 3 min and dehydrated. Finally, DAPI- containing mounting media (Vector Laboratories) was used as contrast staining. Samples were digitized at 20× objective using the Pannoramic 250 scanner (3DHistech) and the number of positive vascular deposits were manually determined in the selected area. Data are expressed as the number of Aβ-positive deposits per area (pixels2). Images were captured using Case Viewer Software (3DHistech).
6
Assay of Estrogenic Compounds
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17β-Estradiol (E2), salicylamide (2-hydroxybenzamide), resorufin (7-hydroxy-3H-phenoxazin-3-one) and resorufin ethyl ether (7-ethoxy-3H-phenoxazin-3-one) were purchased from Sigma-Aldrich (Milan, Italy). G-1 (1-[4-(-6-bromobenzol [1 (link),3 (link)]diodo-5-yl)-3a,4,5,9b-tetrahidro3H5cyclopenta[c]quinolin-8yl]-ethanone), G-15 (3aS,4R,9bR)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinolone and TMS 1-[2,(3,5-Dimethoxyphenyl)ethenyl]-2,4-dimethoxybenzene were obtained from Tocris Bioscience (Space, Milan, Italy). Tyrphostin AG1478 (AG) and PD98059 (PD) were obtained from Calbiochem (DBA, Milan, Italy). All the aforementioned compounds were dissolved in dimethyl sulfoxide (DMSO), except for salicylamide that was dissolved in methanol.
7
Colorimetric Detection of Reactive Oxygen Species
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Amplex Red
(AR) and resorufin were purchased from Sigma-Aldrich (St. Louis, MO).
Hydrogen peroxide (H2O2) and cupric nitrate
(Cu (NO3)2) were obtained from Sinopharm Chemical
Reagent Co., Ltd. The other chemicals (Cr(NO3)3·9H2O, ZnSO4, (CH3COO)2Ca, (CH3COO)2Mg, CdCl2, Pb(NO3)2, FeCl3, Hg(NO3)2) were purchased from Aladdin (Shanghai, China). All the chemicals
were analytical reagent grade and used as received without further
purification. Ultrapure water used throughout was prepared by the
Milli-Q ultrapure water system (18.2 MΩ·cm–1, Millipore System Inc.)
(AR) and resorufin were purchased from Sigma-Aldrich (St. Louis, MO).
Hydrogen peroxide (H2O2) and cupric nitrate
(Cu (NO3)2) were obtained from Sinopharm Chemical
Reagent Co., Ltd. The other chemicals (Cr(NO3)3·9H2O, ZnSO4, (CH3COO)2Ca, (CH3COO)2Mg, CdCl2, Pb(NO3)2, FeCl3, Hg(NO3)2) were purchased from Aladdin (Shanghai, China). All the chemicals
were analytical reagent grade and used as received without further
purification. Ultrapure water used throughout was prepared by the
Milli-Q ultrapure water system (18.2 MΩ·cm–1, Millipore System Inc.)
8
Inhibition of CYP51 by n-Hexane Fraction
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The n-hexane fraction
of Nephthea was screened for its inhibitory
activity against lanosterol 14α-demethylase (CYP51) in comparison
with the drug fluconazole as a reference at the Confirmatory Diagnostic
Unit VACSERA, Cairo, Egypt. 7-EthoxyResorufin (7-ER) is a fluorescent
substrate and competitive suppressor of cytochrome P450 (CYP) isoform
CYP1A1 (IC50 = 0.1 μM). Upon enzymatic cleavage by
CYP1A1 Resorufin was released and its fluorescence was used to quantify
CYP1A1 activity. Resorufin displays excitation/emission maxima of
λmax 572/580 nm, respectively. Resorufin and 7-ethoxyResorufin
obtained from Sigma-Aldrich (St. Louis, MO, USA) are light-sensitive;
therefore, this procedure should be carried out under yellow light
to protect the integrity of the stock solutions. Incubations were
prepared in a black 96-well plate, consisting of a substrate (7ER)
and CaCYP51 bactosomes in 1pH 7.4 00 mM potassium phosphate buffer
containing 5 mM magnesium chloride. Reactions were initiated by adding
40 μL of a 5x NADPH generating system (this can be omitted from
wells containing blanks and standards). The formation of Resorufin
was measured fluorometrically every 30 s through the use of detection
wavelengths (excitation/emission at 572/604 nm) chosen to minimize
interference from NADPH and 7ER. The substrate 7-ethoxyResorufin and
its metabolite Resorufin are both available from Cypex.34 (link)
of Nephthea was screened for its inhibitory
activity against lanosterol 14α-demethylase (CYP51) in comparison
with the drug fluconazole as a reference at the Confirmatory Diagnostic
Unit VACSERA, Cairo, Egypt. 7-EthoxyResorufin (7-ER) is a fluorescent
substrate and competitive suppressor of cytochrome P450 (CYP) isoform
CYP1A1 (IC50 = 0.1 μM). Upon enzymatic cleavage by
CYP1A1 Resorufin was released and its fluorescence was used to quantify
CYP1A1 activity. Resorufin displays excitation/emission maxima of
λmax 572/580 nm, respectively. Resorufin and 7-ethoxyResorufin
obtained from Sigma-Aldrich (St. Louis, MO, USA) are light-sensitive;
therefore, this procedure should be carried out under yellow light
to protect the integrity of the stock solutions. Incubations were
prepared in a black 96-well plate, consisting of a substrate (7ER)
and CaCYP51 bactosomes in 1pH 7.4 00 mM potassium phosphate buffer
containing 5 mM magnesium chloride. Reactions were initiated by adding
40 μL of a 5x NADPH generating system (this can be omitted from
wells containing blanks and standards). The formation of Resorufin
was measured fluorometrically every 30 s through the use of detection
wavelengths (excitation/emission at 572/604 nm) chosen to minimize
interference from NADPH and 7ER. The substrate 7-ethoxyResorufin and
its metabolite Resorufin are both available from Cypex.34 (link)
9
Synthesis and Analysis of Organic Compounds
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The following chemicals, gases,
and materials were used: pyridine (C5H5N, Merck,
EMSURE), methanol (Acros, HPLC grade, 99.99% pure), N2 (Linde,
99.998%), He (Linde, >99%), Ar (Linde, 99.998%), methylcellulose
(Sigma-Aldrich,
4000 CP), acetic acid (Sigma-Aldrich, 99.5%), boehmite (CATAPAL D,
SASOL), zeolite HZSM-5 (ACS material, MR 38), N,N′-bis(2,6-dimethyl phenyl)-perylene-3,4,9,10-tetracarboxylic
diimide (PDI, Sigma-Aldrich), 20/45/100 nm probes (FluoSpheres, carboxylate-modified
microspheres in Milli-Q water, ThermoFisher Scientific), and 7-hydroxy-3H-phenoxazin-3-one (resorufin, Sigma-Aldrich).
and materials were used: pyridine (C5H5N, Merck,
EMSURE), methanol (Acros, HPLC grade, 99.99% pure), N2 (Linde,
99.998%), He (Linde, >99%), Ar (Linde, 99.998%), methylcellulose
(Sigma-Aldrich,
4000 CP), acetic acid (Sigma-Aldrich, 99.5%), boehmite (CATAPAL D,
SASOL), zeolite HZSM-5 (ACS material, MR 38), N,N′-bis(2,6-dimethyl phenyl)-perylene-3,4,9,10-tetracarboxylic
diimide (PDI, Sigma-Aldrich), 20/45/100 nm probes (FluoSpheres, carboxylate-modified
microspheres in Milli-Q water, ThermoFisher Scientific), and 7-hydroxy-3H-phenoxazin-3-one (resorufin, Sigma-Aldrich).
10
Hepatocarcinogenesis Biomarker Analysis
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Diethylnitrosamine (DEN), diaminobenzidene (DAB), gallic acid and resorufin were purchased from Sigma Aldrich (St. Louis, MO, USA). The antibodies of rabbit polyclonal GST-placental form (GST-P), rabbit monoclonal fatty acid synthase (FAS) and mouse monoclonal proliferating cell nuclear antigen (PCNA) antibodies were acquired from MBL (Nagoya, Japan), Cell Signaling Technology (Danvers, MA, USA) and BioLegend (San Diego, CA, USA), respectively. EnvisionTM G/2 Doublestain System with Rabbit/Mouse (DAB+/Permanent Red) was obtained from Dako (Glostrup, Denmark). Avidin–biotin–horseradish peroxidase complex (ABC) kit was acquired from Vector Laboratories (Burlingame, CA, USA). ApopTag peroxidase in situ Apoptosis Detection Kit was obtained from Merck (Kenilworth, NJ, USA). Triglyceride kit assay was received from HUMAN Diagnostics Worldwide (Wiesbaden, Germany). β-Nicotinamide adenine dinucleotide phosphate (NADPH) was supplied by Oriental Yeast Co., Ltd. (Tokyo, Japan).
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