Flp‐In HEK293T‐REx cells were transfected with pcDNA5/FRT/TO AFG3L2E408FLAG. After transfection cells were selected with 100 μg/ml hygromycin (Roth, Cat# CP13.4) for 7 days. Single colonies were selected and expression of the gene of interest was induced with 1 μg/ml tetracycline (Roth, Cat# 0237.1) for 24 h before testing expression via immunoblotting.
Tetracycline
Manufactured by Carl Roth
Sourced in Germany
Tetracycline is a broad-spectrum antibiotic that inhibits bacterial protein synthesis. It is commonly used in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Doxycycline, by Merck Group (3 mentions)
Fetal calf serum (fcs), by Merck Group (3 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (3 mentions)
Gentamicin, by Carl Roth (2 mentions)
Chloramphenicol, by Carl Roth (2 mentions)
Kanamycin, by Carl Roth (2 mentions)
Hygromycin, by Carl Roth (1 mentions)
Streptomycin, by Sarstedt (1 mentions)
96 well plate, by Sarstedt (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Vitek 2 system, by bioMérieux (1 mentions)
Erythromycin, by Carl Roth (1 mentions)
Mueller hinton broth (mhb), by Merck Group (1 mentions)
Ampicillin, by Carl Roth (1 mentions)
Streptomycin, by Carl Roth (1 mentions)
Potato dextrose agar (pda), by Carl Roth (1 mentions)
Ceftriaxone, by Merck Group (1 mentions)
Camhb, by BD (1 mentions)
Dimethyl sulfoxide (dmso), by Cayman Chemical (1 mentions)
Lb medium, by Carl Roth (1 mentions)
12 protocols using tetracycline
1
Inducible AFG3L2 Expression in HEK293T-REx
2
Stable Transfection of NTCP in HEK293 and HepG2 Cells
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Human embryonic kidney (HEK293) cells were stably transfected with human NTCP, C-terminally tagged with the FLAG epitope (further referred to as NTCP-HEK293 cells) as reported before51 (link). Cells were maintained at 37 °C, 5% CO2 and 95% humidity in DMEM/F-12 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA), 4 mM L-glutamine (PAA, Cölbe, Germany) and penicillin/streptomycin (PAA). HepG2 cells stably transfected with NTCP-FLAG (further referred to as NTCP-HepG210 (link)) were cultured under the same conditions in DMEM with all supplements listed above, except for L-glutamine. For induction of the transgene, the medium was supplemented with 1 µg/ml tetracycline (Roth, Karlsruhe, Germany) in the case of the NTCP-HEK293 cells or with 2 µg/ml doxycycline (Sigma-Aldrich) in the case of the NTCP-HepG2 cells.
3
Phenotypic Antimicrobial Susceptibility Testing
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Phenotypic antimicrobial susceptibility testing was performed in collaboration with the IMD Laboratory Greifswald (Germany) using the automated VITEK 2 system (bioMérieux, Marcy l’Etoile, France). Testing was performed using the AST-N389 card, according to the manufacturer’s instructions. MIC values of gentamicin (Carl Roth, Karlsruhe, Germany), streptomycin (Sigma-Aldrich, St. Louis, MO, USA), tetracycline (Carl Roth, Karlsruhe, Germany), and tellurite (Alfa Aesar, Haverhill, MA, USA) were determined by broth microdilution according to ISO Standard 20776-1 [72 ]. Briefly, several single colonies were resuspended in a 0.9% (w/v) NaCl solution until the corresponding suspensions had an OD600 equal to 0.5 McFarland standard turbidity. The bacterial suspensions were then diluted 1:230 in cation-adjusted Mueller–Hinton broth 2 (MH-2; Sigma-Aldrich, St. Louis, MO, USA), corresponding to approximately 105 CFU mL−1. Serial 2-fold dilutions were performed in a 96-well plate (Sarstedt, Nümbrecht, Germany) with concentrations ranging from 64 to 0.5 µg mL−1 for the antibiotics gentamicin, streptomycin, and tetracycline, and from 512 to 0.5 µg mL−1 for the heavy metal tellurite, respectively. Finally, the bacterial suspensions were added and incubated at 37 °C for 18 ± 2 h. The MIC represents the lowest concentration of antimicrobial agent that inhibits visible bacterial growth.
4
Generating Symbiont-free Paramecium Lines
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The antibiotic treatment was performed in order to obtain genetically identical symbiont-free lines from the infected ones. Symbiont-free cells were obtained from both, P. pentaurelia YE9 and P. primaurelia LgJac, through antibiotic treatment (YE9AB and LgJacAB, respectively). Therefore, approximately 30 cells of the stock cultures were transferred to 500 μl of tetracycline (Carl Roth, Karlsruhe, Germany) solution (130 μg ml–1) and incubated for 24 h at 20°C. Later, the cells were washed four times. Individual cells were incubated again in tetracycline solution at 20°C for 24–48 h and then transferred to CM inoculated with R. planticola as food organism. After some rounds of cell division, single cells were treated again with tetracycline for 24 h and subsequently transferred into bacterized CM. The success of the antibiotic treatment was confirmed by FISH.
5
Antibiotic Susceptibility of Campylobacter Strains
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The susceptibility of the isolated Campylobacter strains to selected antibiotics was tested by determining the minimum inhibitory concentrations (MIC) in Mueller Hinton broth (Sigma‐Aldrich). MICs were determined by the broth microdilution procedure in 96‐well flat‐bottomed microtiter plates according to Andrews (2001 ). The antibiotics tested were ciprofloxacin (CIP; Fluka), amoxicillin (AML; Fluka), ampicillin (AMP; Roth, Poland), gentamicin (CN; Roth), streptomycin (S; Roth), tetracycline (TE; Roth), erythromycin (E; Roth), and chloramphenicol (C; Roth). The concentrations for the antibiotics ranged from 0.5 to 256 µg/ml for tetracycline and erythromycin; from 0.25 to 128 µg/ml for amoxicillin, ampicillin, chloramphenicol, erythromycin, and gentamicin; and from 0.125 to 64 µg/ml for ciprofloxacin. The plates were incubated for 24 hr at 42°C in microaerophilic conditions. A C. jejuni reference strain (ATCC 33560) was used as a control. All tests were run in triplicate. The Campylobacter spp. isolates were classified as resistant or susceptible according to EUCAST, European Committee on Antimicrobial Susceptibility Testing 2016a , 2016b .
6
Isolation and Cultivation of Colletotrichum from Lupin Tissues
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Symptomatic (dried) lupin stem or pod tissue (Fig. 1 ) of 1–3 cm was surface sterilized (after rehydration in sterile ddH2O for dried samples) for 5 s with 0.25% sodium hypochlorite solution and rinsed thrice for 5 s in sterile ddH2O. Thin slices of 1 mm were cut and placed on PDA (potato dextrose agar, Carl Roth, Karlsruhe, Germany) amended with Tetracycline (0.02 g/l, Carl Roth) for 3 to 4 days at 22 °C in the dark. Single cultures were selected and grown on fresh PDA plates amended with Tetracycline for 4 to 6 days at 22 °C in the dark and suspected Colletotrichum species were sub-cultured. Single spore cultures were obtained and transferred to PDA and maintained at 22 °C in the dark as working cultures and stored at − 80 °C in 25% glycerol for long-term storage.
7
Antimicrobial Susceptibility Testing Protocol
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For the determination of the MIC values of plant extracts, cation adjusted Mueller Hinton II broth was used (CAMHB, Becton Dickinson). Dimethyl sulfoxide (DMSO, Merck Gesellschaft GmbH, Vienna, Austria) was used as a solvent for the herbal extracts. The broth microdillution method for antimicrobial susceptibility testing was applied in accordance with the CLSI standards [31 ,32 ]; instead of antibiotics, SFE extracts were applied. Ceftriaxone (Sigma Aldrich, city, country) and tetracycline (Carl Roth, Karlsruhe, Germany) were included as controls. The investigated concentrations of plant extracts were 2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5 and 1.25 expressed in μg/mL. Sedanolide (3-butyl-3a,4,5,6-tetrahydro-1(3H)-isobenzofuranone) with a purity of ≥95%, and (+)-Limonene (1-methyl-4R-(1-methylethenyl)-cyclohexene) with a purity of ≥98% (Cayman chemicals, Ann Arbor, MI, USA) were dissolved in DMSO and investigated at the same concentrations as the SFE extracts. The final bacterial inoculum density of 5 × 105 CFU/mL was achieved by adding 5 μL of 1–2 × 107 CFU/mL suspension of the investigated strain in microplate wells with 100 μL of previously added CAMHB. Microplates were incubated for 18–24 h at 37 °C.
8
Stable Transfection of NTCP in HEK293 and HepG2 Cells
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Human embryonic kidney (HEK293) cells were stably transfected with human NTCP and C-terminally tagged with the FLAG epitope (further referred to as NTCP-HEK293 cells) as reported before [23 (link),24 (link)]. Cells were maintained at 37 °C, 5% CO2, and 95% humidity in DMEM/F-12 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA), 4 mM L-glutamine (PAA, Cölbe, Germany), and penicillin/streptomycin (PAA). HepG2 cells stably transfected with NTCP-FLAG (further referred to as NTCP-HepG2 [9 (link)]) were cultured under the same conditions in DMEM with all supplements listed above, except for L-glutamine. For induction of the transgene, the medium was supplemented with 1 µg/mL tetracycline (Roth, Karlsruhe, Germany) in the case of the NTCP-HEK293 cells or with 2 µg/mL doxycycline (Sigma-Aldrich) in the case of the NTCP-HepG2 cells.
9
Stable Transfection of NTCP in HEK293 and HepG2 Cells
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Human embryonic kidney (HEK293) cells were stably transfected with human NTCP, C-terminally tagged with the FLAG epitope (further referred to as NTCP-HEK293 cells) as reported before [19 (link)]. Cells were maintained at 37 °C, 5% CO2, and 95% humidity in DMEM/F-12 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA), 4 mM L-glutamine (PAA, Cölbe, Germany) and penicillin/streptomycin (PAA). HepG2 cells stably transfected with NTCP-FLAG (further referred to as NTCP-HepG2 [10 (link)]) were cultured under the same conditions in DMEM with all supplements listed above, except for L-glutamine. For induction of the transgene, the medium was supplemented with 1 µg/mL tetracycline (Roth, Karlsruhe, Germany) in the case of the NTCP-HEK293 cells or with 2 µg/mL doxycycline (Sigma-Aldrich) in the case of the NTCP-HepG2 cells.
10
Cultivation and Induction of Fluorescent Bacterial Strains
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Two strains were used in the experiments: P. aeruginosa PAO1 pVLT31-eGFP and E. coli Nissle 1917 pVLT31-eGFP. For the cultivation of each bacterial strain, precultures were prepared in 5 mL of LB medium (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) containing 10 µg mL−1 of tetracycline (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). These precultures were incubated at 37 °C for 16 h while shaking at 150 rpm. The next day, the OD600 values of the precultures were determined, and a flask containing 25 mL LB medium and 10 µg mL−1 tetracycline was subsequently inoculated with an initial OD600 of 0.05. Cultivation of the flasks was performed at 37 °C with shaking (150 rpm). Upon reaching an OD600 of 0.6, cultures were induced with 0.4 mM isopropyl-ß-d-1-thiogalactopyranoside (IPTG, Carl Roth GmbH + Co. KG, Karlsruhe, Germany). The bacterial cultures were then incubated for up to three more hours until the stationary phase was reached. In order to achieve the desired cell numbers per mL in the subsequent experiments, the cells were counted under the microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany) using a Thoma cell counting chamber (LO-Laboroptik GmbH, Friedrichsdorf, Germany).
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