All ELISA kits were based on the sandwich ELISA method. TCs were washed with PBS when reached 70–80% confluence and covered with serum-free media for 24 h. Cell culture supernatants were harvested, centrifuged to avoid cell debris in the sample, transferred to liquid nitrogen, and stored at -80 °C. Thawed samples were analyzed to quantify chemokine secretion using ELISA kits from R&D Systems (CCL2, CCL5, CXCL1, CXCL2, CXCL10, CXCL17) according to the manufacturer’s instructions. To examine chemokine secretion from TCs after STAT inhibition, fludarabine or nifuroxazide (Sigma) were added to serum-free media at 50 µM (24 h) [55 (link), 56 (link)]. As control, the serum-free media was supplemented with 0.1% DMSO.
Nifuroxazide
Manufactured by Merck Group
Sourced in United States
Nifuroxazide is a laboratory chemical compound used in research and development. It is a nitrofuran derivative with antimicrobial properties. Nifuroxazide is commonly used as a reference standard or in the development of analytical methods.
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Lab products found in correlation
Cremophor el, by Merck Group (2 mentions)
Rh123, by Merck Group (2 mentions)
Dcfh da, by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (2 mentions)
N acetyl cysteine (nac), by Beyotime (2 mentions)
Spectinomycin, by Merck Group (2 mentions)
Dihydrostreptomycin, by Merck Group (2 mentions)
Multiskan ascent, by Thermo Fisher Scientific (2 mentions)
Lithium chloride (licl), by Merck Group (2 mentions)
Sb 216763, by Adipogen (2 mentions)
Poly l lysin, by Harvard Bioscience (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Fludarabine, by Merck Group (1 mentions)
Cd11b fitc, by BD (1 mentions)
Hoechst33358, by Beyotime (1 mentions)
Mitosox red, by Yeasen (1 mentions)
Stat3 p stat3tyr705, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti ki 67, by Merck Group (1 mentions)
14 protocols using nifuroxazide
1
ELISA-Based Chemokine Secretion Analysis
2
Nifuroxazide and SAT05f Inhibitor Study
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Eight-week-old male C57BL/6 and BALB/c mice were purchased from Experimental Animal Center of Zhengzhou University (Zhengzhou, Henan, China). Nifuroxazide was obtained from Sigma (St. Louis, MO, USA), and was dissolved in DMSO. SAT05f, an inhibitory ODN used in this and previous studies, has a sequence of 5′-CCTCCTCCTCCTCCTCCTCCTCCT-3′ and was provided by Sangon Biotech (Shanghai, China).
3
Nifuroxazide Synthesis and Characterization
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MTT, DMSO, DCFH-DA, Rh123, Cremophor EL and PI were purchased from Sigma Chemical Co. (St Louis, MO, USA). NAC and Hoechst33358 were obtained from Beyotime (Beijing, China). MitoSOX red and Amplex red were purchased from Yeasen (Shanghai, China) and Invitrogen (Carlsbad, USA), respectively. The Annexin V-FITC apoptosis detection kit was purchased from KeyGen Biotech (Nanjing, China). The primary antibodies were obtained from Cell Signaling Technology Company (Beverly, MA). FITC-CD11b, and PE-Gr1 conjugated antibodies were acquired from BD Biosciences.
Nifuroxazide was purchased from Xiyashiji Chemical Co. Ltd (Chengdu, Sichuan, China), and was determined by 1H-NMR, 13C-NMR and ESI-MS analysis. For the in vitro studies, Nifuroxazide was prepared in DMSO at a stock concentration of 20 mM and diluted in the relevant medium at final DMSO concentration of 0.1% (V/V), and medium with 0.1% DMSO served as vehicle control. For in vivo experiment, Nifuroxazide was dissolved in 25% (v/v) Cremophor EL/ethanol (50:50, Sigma Cremophor EL, 100% ethyl alcohol) and 75% ultrapure water.
Nifuroxazide was purchased from Xiyashiji Chemical Co. Ltd (Chengdu, Sichuan, China), and was determined by 1H-NMR, 13C-NMR and ESI-MS analysis. For the in vitro studies, Nifuroxazide was prepared in DMSO at a stock concentration of 20 mM and diluted in the relevant medium at final DMSO concentration of 0.1% (V/V), and medium with 0.1% DMSO served as vehicle control. For in vivo experiment, Nifuroxazide was dissolved in 25% (v/v) Cremophor EL/ethanol (50:50, Sigma Cremophor EL, 100% ethyl alcohol) and 75% ultrapure water.
4
Nifuroxazide Compound Characterization and Evaluation
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Nifuroxazide was purchased from Xiyashiji Chemical Co. Ltd (Chengdu, Sichuan, China), and was measured by 1H-NMR, 13C-NMR and ESI-MS analysis. For the in vitro assays, Nifuroxazide was prepared in dimethyl sulfoxide (DMSO) at a stock concentration of 20 mM and stored at −20 °C. Then, it was diluted in the relevant medium at a final DMSO concentration of 0.1% (v/v), and the medium with 0.1% DMSO served as a vehicle control. For in vivo studies, Nifuroxazide was dissolved in 25% (v/v) Cremophor EL/ethanol (50 : 50, Sigma Cremophor EL, 100% ethyl alcohol) and 75% ultrapure water.
DMSO, MTT, DCFH-DA, Rh123 and Cremophor EL were from Sigma Chemical Co. (St. Louis, MO, USA). Hoechst 33258 and NAC were obtained from Beyotime (Beijing, China). The primary antibodies against Stat3/p-Stat3Tyr705, MMP-2, MMP-9, cleaved caspase-3, Bax, Bcl-2 and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). FITC-CD11b-, PE-Gr1-, FITC-CD8a-, PE-CD69-, PE-CD206- and APC-F4/80-conjugated antibodies were obtained from BD Biosciences (San Diego, CA, USA). Anti-Ki-67 mouse monoclonal was purchased from Merck Millipore (Billerica, MA, USA). The Annexin V-FITC Apoptosis Detection Kit was obtained from KeyGen Biotech (Nanjing, China).
DMSO, MTT, DCFH-DA, Rh123 and Cremophor EL were from Sigma Chemical Co. (St. Louis, MO, USA). Hoechst 33258 and NAC were obtained from Beyotime (Beijing, China). The primary antibodies against Stat3/p-Stat3Tyr705, MMP-2, MMP-9, cleaved caspase-3, Bax, Bcl-2 and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). FITC-CD11b-, PE-Gr1-, FITC-CD8a-, PE-CD69-, PE-CD206- and APC-F4/80-conjugated antibodies were obtained from BD Biosciences (San Diego, CA, USA). Anti-Ki-67 mouse monoclonal was purchased from Merck Millipore (Billerica, MA, USA). The Annexin V-FITC Apoptosis Detection Kit was obtained from KeyGen Biotech (Nanjing, China).
5
Nifuroxazide-loaded PEGCE Nanoparticles
6
Analytical Methodology for ABZ-SO and TZO
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ABZ-SO and TZO standards used for the analytical methodology were synthesized by Drs. Rafael Castillo-Bocanegra and Alicia Hernández Campos at the Facultad de Química, UNAM, Mexico. Chemical identity was confirmed by NMR, MS analysis, and melting point determination. ABZ, mebendazole (MBZ), and nifuroxazide (NFZ), used as internal standards (IS) for plasma ABZ-SO and plasma TZO analysis, respectively, were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Methanol, acetonitrile, ether, dichloromethane, and chloroform were of HPLC grade (Mallinckrodt Co., St. Louis, MO, USA). Formic acid (Sigma-Aldrich Co., St. Louis, MO, USA) was of analytical reagent-grade. Water was obtained from a Milli-Q Water System (Millipore Corporation, Bedford, MA, USA).
For the pharmacokinetic studies, ABZ suspension (Zentel®, 400 mg/10 mL; Glaxo SmithKline) and NTZ suspension (Paramix®, 100 mg/5 mL; Liomont Laboratories) were used.
For the pharmacokinetic studies, ABZ suspension (Zentel®, 400 mg/10 mL; Glaxo SmithKline) and NTZ suspension (Paramix®, 100 mg/5 mL; Liomont Laboratories) were used.
7
Antibiotic Susceptibility Assay of Mechanosensitive Ion Channels
8
Nifuroxazide Treatment in BALB/c Mice
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The recipient mice were 8–12-week-old BALB/c male mice (n=20 per group), and the donor mice were 8–12-week-old C57BL/6 male mice. All mice were obtained from the Experimental Animal Center of Zhengzhou University (Zhengzhou, China) and were fed for about one week to adapt to the environment. Subsequently, in the next week, the recipient mice were fed with sterile food and acidified water. All experiments were performed according to the Institutional Animal Care and Use Committee Guidelines and were approved by the Ethics Committee of Xinxiang Medical University (Xinxiang, China). The nifuroxazide used was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) and dissolved in dimethylsulfoxide.
9
Antibiotic Susceptibility Assay of Mechanosensitive Ion Channels
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E.coli strain MJF612 was used as a host for E.coli MscL (MscL), K55T E.coli MscL (K55T MscL), Bacillus subtilis MscL (Bsub MscL), Staphylococcus aureus MscL (Sau MscL) and E.coli MscS (MscS) constructs in the pB10d expression vector. Overnight cultures were grown in CphM plus 100µg ml−1 ampicillin in a shaker incubator at 37°C and rotated at 250 cycles per min. The next morning cultures were diluted to 1:60 in the same media and grown for 30 minutes before inducing expression with 1mM IPTG. After 1 hour of induction all cultures were adjusted to an OD600 of 0.1. The cultures were then diluted 1:3 into pre-warmed CphM plus 100µg ml−1 ampicillin plus the antibiotic being tested. Cultures were loaded in 96 well plates (180µl/ well) in 7 replicates; plates were sealed with a breathable film for 96 well plates to avoid evaporation, wrapped in foil and incubated at 37°C for 16 hours without shaking. The OD600 of the cultures was measured using a Multiskan Ascent (Thermo Scientific Inc., Waltham, MA, USA). The antibiotics were diluted to their final concentration from the following stock solutions: viomycin sulfate, (USP, Rockville MD) 5mM in H2O; nifuroxazide (Sigma, St Louis, MO) 5mM in DMSO; dihydrostreptomycin (Sigma, St Louis, MO) and spectinomycin (Sigma, St Louis, MO) 50mM in H2O.
10
Nifuroxazide Cytotoxicity on Cancer Cells
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The Nifuroxazide used in this study was obtained from Sigma-Aldrich (USA). The HepG2, Huh7, and H22 cell lines were maintained at Xinxiang Key Laboratory of Tumor Vaccine and Immunotherapy, located at Xinxiang Medical University in Xinxiang, Henan (P.R. China).
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