Dimethyl sulfoxide (DMSO), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, quercetin, 5′-fluorodeoxyuridine (FUdR), ampicillin and epigallocatechin gallate (EGCG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gallic acid was purchased from TCI America (Portland, OR, USA), L-ascorbic acid from Calbiochem (San Diego, CA, USA), 2,7-dichlorofluorescein diacetate (H2DCF-DA) from Fluka Chemie GmbH (Buchs, Switzerland), juglone (5-hydroxy-1,4-naphthalenedione) from Sigma-Aldrich GmbH (Steinheim, Germany), and sodium azide from AppliChem GmbH (Darmstadt, Germany). Other reagents used in the extraction process were of analytical grade and purchased from RCI Labscan (Bangkok, Thailand).
Sodium azide
Manufactured by AppliChem
Sourced in Germany
Sodium azide is a chemical compound with the formula NaN3. It is a colorless crystalline solid that is commonly used as a laboratory reagent. Sodium azide has a high reactivity and is typically handled with caution due to its potential toxicity.
Automatically generated - may contain errors
Lab products found in correlation
Folin ciocalteu reagent, by Merck Group (4 mentions)
Epigallocatechin gallate (egcg), by Merck Group (4 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (4 mentions)
Quercetin, by Merck Group (3 mentions)
Gallic acid, by Tokyo Chemical Industry (3 mentions)
Juglone 5 hydroxy 1 4 naphthalenedione, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Juglone, by Merck Group (2 mentions)
Cm h2dcfda, by Merck Group (2 mentions)
Biorevo bz 9000 fluorescence microscope, by Keyence (2 mentions)
L glutamic acid, by Merck Group (2 mentions)
2 2 azino bis 3 ethylbenzothiazoline 6 sulfonic acid diammonium salt, by Merck Group (1 mentions)
Pronase e, by Merck Group (1 mentions)
Calcium chloride, by Merck Group (1 mentions)
Ascorbic acid, by AppliChem (1 mentions)
Fluorescence microscope, by Keyence (1 mentions)
Leupeptin, by AppliChem (1 mentions)
Dimethyl sulfoxide dmso 2 2 azino bis 3 ethylbenzothiazoline 6 sulfonic acid diammonium salt abts, by Merck Group (1 mentions)
2 7 dichlorofluorescein diacetate h2dcf da, by Merck Group (1 mentions)
H2dcfda, by Merck Group (1 mentions)
14 protocols using sodium azide
1
Antioxidant Evaluation of Phytochemicals
2
Immunolabeling of Drosophila Synaptic Proteins
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Several CRG whole-mounts were incubated with an antibody directed against the Drosophila synaptic protein synapsin [39 (link)]. To facilitate the antibody penetration, the ganglion sheath was treated for 30 s with small crystals of a proteolytic enzyme (Pronase E, Merck, Darmstadt, Germany). The enzyme was washed out by repeated rinsing with stick insect saline. Then, the detached sheath was manually removed with fine forceps and a pair of sharp scissors. Specimens were then fixed and washed as described above. Afterwards, ganglia were preincubated in 0.1 M PBS containing 5% normal goat serum (S1000, Linaris, Wertheim, Germany), 1% Triton-X 100 (Fluka, Buchs, Switzerland) and 0.1% sodium azide (AppliChem) for 1–2 hours to block unspecific antibody binding. The same solution was used for washes, and the dilution of primary and secondary antibodies. The monoclonal mouse anti-synapsin antibody SYNORF1 (kindly provided by Erich Buchner, University of Würzburg, Germany) was applied at a dilution of 1:25 for 2 x 3 days at 4°C. Subsequently, three washes of 2 hours each were carried out and ganglia were incubated with DyLight 633-conjugated goat anti-mouse IgG antibody (ThermoScientific) diluted 1:100 in blocking-solution for 2.5 days at 4°C. Ganglia were washed 3 x 1 hour in PBS, dehydrated and cleared as described above.
3
Preparation of Hashemi Rice-Based Products
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Hashemi rice and rice flour were obtained from a local market (Hashemi variety, Rudsar, Iran). Xanthan gum and sodium alginate were purchased from Fufeng Company (Fufeng Company). Low acyl gellan was purchased from Qiaoli Co. (Qiaoli Co.). Sodium azide was obtained from AppliChem GmbH Co. (AppliChem GmbH Co.). Calcium chloride was obtained from Merck Co. (Merck Co.). Sugar, sodium chloride salt, monoglyceride, and corn starch were bought from a local market (Mashhad, Iran).
4
Antioxidant Capacity Measurement Assay
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Juglone (5-hydroxy-1,4-naphthalenedione), EGCG, and DPPH● were purchased from Sigma-Aldrich GmbH (Darmstadt, Germany), and sodium azide and ascorbic acid were purchased from AppliChem GmbH (Darmstadt, Germany).
5
Antioxidant Assay Protocol
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Purified EGCG from green tea (purity ≥ 95%), 2,7-dichlorofluorescein diacetate (DCF-DA), 2,2-diphenyl-1-picrylhydrazyl (DPPH•), butylated hydroxyl anisole (BHA), bis (neocuproine) copper (II) chelate (CUPRAC reagent), vitamin C (Vit C) and juglone (5-hydroxy-1,4-naphthalenedione) were purchased from Sigma-Aldrich, Germany. Sodium azide was bought from AppliChem GmbH, Darmstadt, Germany; Folin-Ciocalteu reagent from Merck, Darmstadt, Germany.
6
Oxidative Stress Response in Aged Nematodes
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Age synchronized N2 worms (L1 stage, grown in S-medium) were sorted into groups and treated with GE or AlkE for 48 h, except the control group. Afterwards, the worms were washed with M9 buffer and incubated with 1 mL 50 μM CM-H2DCFDA solution (1 h at 20 °C) (6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate; Fluka Chemie GmbH, Buchs, Switzerland), washed again with M9 and mounted on a glass slide, and a drop of 10 mM sodium azide (AppliChem GmbH, Darmstadt, Germany) was added to paralyze the nematodes. Live images were captured from at least 30 worms per group, using a fluorescence microscope (Keyence Deutschland GmbH, Neu-Isenburg, Germany; λEx 480/20 nm; λEm 510/38 nm). The relative fluorescence of the whole body was determined densitometrically using ImageJ software version 1.48 (National Institutes of Health, Bethesda, MD, USA). The results are presented as mean fluorescence intensity (mean ± SEM) and were compared by one-way ANOVA followed by Bonferroni (post-hoc). The assay was repeated three times.
7
Caffeine, Theophylline, and Theobromine Effects on ROS in C. elegans
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Synchronized L1 larval worms (N2 grown in S-medium) were treated with 1 mM caffeine, 5 mM caffeine, 1 mM theophylline, 5 mM theophylline, and 1 mM theobromine (due to lower solubility of theobromine, 5 mM theobromine could not be obtained) for 48 h, while a control group was left untreated. All groups were then treated with 50 μM CM-H2DCFDA (Sigma-Aldrich GmbH, Steinheim, Germany) and incubated in the dark for 1 h at 20 °C. Worms were washed once with M9 buffer to remove any excess dye and then mounted on a glass slide with a drop of 10 mM sodium azide (AppliChem GmbH, Darmstadt, Germany) for paralysis. CM-H2DCFDA is taken up by the cells and then oxidized by ROS into a fluorescent dye (DCF). Then the fluorescent intensity is related to the ROS level in C. elegans. Using a BIOREVO BZ-9000 fluorescence microscope (Keyence Deutschland GmbH, Neu-Isenburg, Germany) equipped with a mercury lamp, the slides were photographed (λex 480/20 nm, λem 510/38 nm, a 10× bjective lens, a constant exposure time) and analyzed. The relative fluorescence of the whole body was determined densitometrically using ImageJ (National Institutes of Health, Bethesda, MD). The results are presented as the percent mean fluorescent intensity compared to the untreated control. All the experiments were carried out at least three times and compared by one-way ANOVA followed by Dunnett’s method.
8
Isolation of Nanovesicles from Tomato Fruits
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Nanovesicles were isolated from Piccadilly variety tomato fruits (200 g, Vittoria Colonna s.r.l., Sicily, Italy) by differential ultracentrifugation (dUC) according to Bokka et al. [1 (link)]. In brief, after washing and removing exocarp, tomatoes were homogenized in a kitchen mixer homogenizer by 3 cycles of 10 s in extraction buffer composed of 100 mM phosphate, 10 mM ethylenediaminetetraacetic acid (EDTA) (J.T. Baker, Deventer, The Netherlands) (pH 8, 170 mL) containing protease inhibitors 1 mg/mL leupeptin (0.085 mL, AppliChem, Darmstadt, Germany), 100 mM phenylmethylsulfonyl fluoride (PMSF, 0.425 mL) and 1 M sodium azide (0.272 mL) AppliChem, Darmstadt, Germany). The isolation of NVs were performed using four low-velocity centrifugation steps at increasing centrifugal force, i.e., 400× g, 800× g, 2000× g and 15,000× g each of them for 30 min at 22 °C. Supernatant from the last low-velocity centrifugation step was centrifuged at 100,000× g for 2 h at 4 °C using an SW28Ti rotor in Beckman Coulter Optima L-90K ultracentrifuge (Beckman Coulter, Brea, CA, USA). The pellet containing the NVs was re-suspended in a small volume of buffer.
9
Fluorescent Worm Imaging Protocol
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To analyze the effect of different treatments on the expression of the fluorescent reporter GFP (green fluorescent protein) and the levels of DCF (2',7'-dichlorofluorescein), living worms were mounted onto a glass slide with a drop of 10 mM sodium azide (AppliChem GmbH, Darmstadt, Germany). Fluorescence images (λex 488 nm and λem 540) of at least 30 worms/treatment were acquired at constant exposure time for each experiment, using a Keyence Biorevo BZ-9000 fluorescence microscope (Keyence Deutschland GmbH, Neu-Isenburg, Germany). The fluorescence intensity was densitometrically determined in mean pixels using ImageJ version 1.52 (National Institutes of Health, Bethesda, MD, USA), an open source image processing and analysis software.
10
Antioxidant Compound Characterization Protocol
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Juglone (5-hydroxy-1,4-naphthoquinone) and 2,7-dichlorofluorescein diacetate (H2DCF-DA) were obtained from Sigma-Aldrich GmbH (Steinheim, Germany). Sodium azide was obtained from AppliChem GmbH (Darmstadt, Germany). EGCG (purity ≥ 95%) was purchased from Sigma-Aldrich (München, Germany). Dimethyl sulfoxide (DMSO), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, and quercetin were obtained from Sigma-Aldrich (MO, USA). Gallic acid was purchased from TCI America (OR, USA). L-Ascorbic acid was obtained from Calbiochem (CA, USA). Other reagents used for extraction were of analytical grade and were purchased from RCI Labscan (Bangkok, Thailand).
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