LB medium (5 g yeast extract, 10 g trypton, 5 g NaCl/ l); M9 medium (64 g Na2HPO4.7H2O, 15 g KH2PO4, 2.5 g NaCl, 5 g NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2, 0.4% glucose/l); PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4); S15 and L1 specific antisera, raised in sheep were obtained from Dr. Nierhaus. Horseradish peroxidase (HRP)-conjugated rabbit anti-sheep (CodeNo: 313-035-003; LotNo: 106383) and donkey anti-rabbit (CodeNo: 711-035-152; LotNo: 103871) secondary antibodies were from Jackson ImmunoResearch. HRP-substrate: for detection a mixture of 1 ml solution A + 100 μl solution B + 1 μl solution C was freshly prepared (solution A: 0.1 mM TRIS (pH8.6), 25 mg Luminol, 100 ml distilled H20; solution B: 11 mg p-hydroxycoumaric acid, in 10 ml DMSO; solution C: H2O2 (30%). Antibiotics were used in concentrations as indicated: Ampicillin (Applichem-A0839,0100), chloramphenicol (Sigma-C0378), erythromycin (Sigma-E6367), kanamycin (Roth-T832.4) and neomycin (Sigma-N1876).
Neomycin
Manufactured by Carl Roth
Sourced in Germany
Neomycin is a broad-spectrum antibiotic that inhibits bacterial protein synthesis. It is commonly used in laboratory settings for selective growth of bacterial cultures.
Automatically generated - may contain errors
Lab products found in correlation
Ampicillin, by AppliChem (1 mentions)
Kanamycin, by Carl Roth (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Donkey anti rabbit, by Jackson ImmunoResearch (1 mentions)
Horseradish peroxidase hrp conjugated rabbit anti sheep, by Jackson ImmunoResearch (1 mentions)
Erythromycin, by Merck Group (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Pmscv, by Takara Bio (1 mentions)
Chloramphenicol, by Carl Roth (1 mentions)
Penicillin g, by Carl Roth (1 mentions)
Polymyxin b, by Carl Roth (1 mentions)
Vancomycin, by Carl Roth (1 mentions)
Tak 242, by Cayman Chemical (1 mentions)
Metronidazole, by Merck Group (1 mentions)
Ampicillin, by Carl Roth (1 mentions)
Zymobiomics dna miniprep kit, by Zymo Research (1 mentions)
5 protocols using neomycin
1
Bacterial Growth Media and Antibody Detection
2
Generation of BCL-2 and Phospho-mutant MCL-1 Cell Lines
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For BCL-2 overexpression, cells were transduced with murine stem cell virus (pMSCV, Clontech, Mountain View, CA) vector containing mouse BCL-2 or empty vector using the packaging cell line 293T (BD Biosciences). Stable cell lines were selected with 10 μg/ml Blasticidin (Invitrogen). For phospho-mutant MCL-1, cells were transduced with a pCMV vector containing 4A (S64A/S121A/S159A/T163A) and empty vector by Genentech (South San Francisco) and were selected with Neomycin (Roth).
3
Antibiotic Treatment for Polyp Microbiome Reduction
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Single native polyps were placed in 48 well multiwell plates in 1 mL ASW supplemented with an antibiotic mixture (Provasoli’s antibiotic mixture with final concentrations of 360,000 U/L penicillin G, 1.5 mg/L chloramphenicol, 1.8 mg/L neomycin, and 9,000 U/L polymyxin B; all components from Carl Roth, Karlsruhe, Germany). No food was provided during the antibiotic treatment. The reduction and consequent change of the microbiota were tested by plating a single homogenized polyp (10 replicates) on Marine Bouillon agar plates (Carl Roth, Karlsruhe, Germany). Plates were incubated for 5 days at 20°C. Colony forming units (cfu) were calculated, and an 87 ± 9% reduction per polyp was determined.
4
Bone Marrow Cell Transplantation
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Lin− cells isolated from bone marrow of B6.SJL-PtprcaPepcb/BoyCrl mice (Ly5.1) were washed 1 day after transduction and resuspended in PBS. 5 × 105 to 1 × 106 cells were transplanted into lethally irradiated mice (9.5 Gy) via tail vain injection. Transplanted mice were kept in individually ventilated cages and drinking water was supplemented with 1.6 g l−1 neomycin (Carl Roth, Karlsruhe, Germany) for 2 weeks.
5
TAK-242 Modulates Microbiota in Colitis
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TAK-242 (Cayman Chemical, via Biomol GmbH, Hamburg, Germany), a small-molecule TLR4 inhibitor that disrupts TLR4 interaction with adaptor molecules TIRAP and TRAM (17 (link)), was applied to BALB/c mice during DSS treatment. Mice were treated with 3 mg/kg TAK-242 i.p. every other day for 4 (acute DSS colitis) to 5 (DSS cycles during CAC induction) injections. To deplete the commensal microbiota, mice were orally treated with an antibiotic cocktail (Abx) including 1 mg vancomycin, 2 mg neomycin, 2 mg ampicillin (all Carl Roth GmbH, Karlsruhe, Germany), and 2 mg metronidazole (Sigma-Aldrich) per day and mouse. Efficient depletion was confirmed by quantification of microbial DNA in feces. In brief, stool samples were collected from mice, the weight was measured and then frozen at -80°C until further analysis. DNA from stool samples was extracted using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Freiburg, Germany) according to the manufacturer´s recommendations.
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