Transvaginal ultrasound ovum pick up (OPU) was performed as previously reported [14 (link), 16 (link)]. Briefly, an Aloka real-time B-mode ultrasound system (Aloka SSD-4000, Hitachi Aloka Medical Ltd., Tokyo, Japan) and a 7.5 MHz electronic convex transducer (Hitachi Aloka Medical Ltd., Tokyo, Japan) were used to visualize the ovaries (S1 Fig ). Visible follicles ≥ 3 mm in diameter were punctured using a disposable 20G x 2 ¾” needle (0.9 x 70 mm, Terumo, Eschborn, Germany). A vacuum pump (Aspirator 3, Labotect GmbH, Göttingen, Germany), adjusted to a negative pressure of 60 mmHg (20 ml/min) was used to recover follicular fluid. After puncture of four to five follicles, the oocyte collection system was flushed with Dulbecco’s PBS medium (AppliChem, Darmstadt, Germany), supplemented with 2.2 IU/ml heparin (AppliChem), 1% newborn calf serum (NBCS; PPA Laboratories, Coelbe, Germany), 6 μg/ml penicillin G (AppliChem) and 50 μg/ml streptomycin sulphate (AppliChem) for the standard protocol (TCM24) or additionally supplemented with 3-isobutyl-1-methylxanthine (IBMX, 500 μM, Sigma-Aldrich) for the protocol using the cAMP regulators (cAMP30) and dimethyl sulfoxide (DMSO, 46.6 mM, Sigma-Aldrich) as vehicle control (DMSO30). The vehicle control was necessary because IBMX was dissolved in DMSO. The interval from ovum pick up to oocyte searching did not exceed 20 min.
Penicillin g
Manufactured by AppliChem
Sourced in Germany
Penicillin G is a chemical compound used in laboratory settings. It is a type of antibiotic that can be used for various research and testing purposes. The core function of Penicillin G is to inhibit the growth of certain bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by AppliChem (2 mentions)
Roswell park memorial institute (rpmi), by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Heparin, by AppliChem (1 mentions)
Streptomycin sulphate, by AppliChem (1 mentions)
3 isobutyl 1 methylxanthine ibmx, by Merck Group (1 mentions)
Dulbecco s pbs medium, by AppliChem (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Silica gel 60, by Carl Roth (1 mentions)
Melting point m 560 apparatus, by Büchi (1 mentions)
3 4 5dimethylthiazol 2 yl 2 5 diphenyl tetrazoliumbromide mtt, by AppliChem (1 mentions)
Autopol 4, by Rudolph Research Analytical (1 mentions)
Silica gel 60, by Merck Group (1 mentions)
Chloramphenicol, by Carl Roth (1 mentions)
Tigecycline, by Merck Group (1 mentions)
Vancomycin, by Merck Group (1 mentions)
Meropenem, by Merck Group (1 mentions)
Spectinomycin, by Carl Roth (1 mentions)
Erythromycin, by Carl Roth (1 mentions)
6 protocols using penicillin g
1
Transvaginal Ultrasound-Guided Oocyte Retrieval
2
Analytical Methods for Chemical Characterization
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All solvents (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Fisher Chemical (Pittsburgh, PA, USA). Streptomycin, penicillin G, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from AppliChem (Darmstadt, Germany). The cell medium (RPMI, acquired from Sigma-Aldrich, USA) used in the experiments consisted of RPMI 1640 with 20 mM HEPES and l -glutamine, without sodium bicarbonate, containing 10% (v/v) fetal bovine serum, 200 mg mL−1 Streptomycin, and 200 IU mL−1 penicillin. Silica gel 60, particle size distribution 0.02–0.045 mm (Carl Roth, Karlsruhe, Germany), was used for column chromatography. Thin-layer chromatography (TLC) was performed on Merck plates (Darmstadt, Germany), layer thickness 0.2 mm with Silica gel 60 and fluorescence indicator F254. Visualization was accomplished by spraying the plates with a mixture of sulfuric and nitric acids (1:1) followed by short, gentle heating. Melting point was determined on a Büchi (Flawil, Switzerland) M-560 melting point apparatus. Optical rotation was measured in chloroform on an Autopol IV (Rudolph Research Analytical, Flanders, NJ, USA) polarimeter equipped with a sodium lamp (589 nm) and a 1 mL cell with a 1 dm path length.
3
Antibiotic Susceptibility Testing Protocol
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Antibiotic MICs were determined by the broth microdilution method according to the CLSI standard guidelines M07-A8, M100-S20, and M45-P (50 , 51 , 53 ). Meropenem, amikacin, gentamicin, linezolid, and tigecycline were purchased from Sigma Aldrich (Germany). Vancomycin and tetracycline were obtained from Merck (Germany). Clindamycin, ciprofloxacin, lincomycin, and penicillin G were obtained from Applichem (Germany). Ampicillin, chloramphenicol, spectinomycin, streptomycin, and erythromycin were purchased from Carl Roth GmbH (Germany). MICs of the quality control strains S. aureus ATCC 29213 and E. coli ATCC 25922 were recorded on each day for all tested antibiotics and fell within the acceptable range prescribed by the CLSI.
4
Inducible Recombination of Gene Libraries
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Experiments were performed using HEK293T landing pad cell line TetBxb1BFPiCasp9 Clone 12, which was characterized previously47 (link). The cells were maintained in Dulbecco’s Modified Eagle´s Medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma), 64.43 mM Penicillin G (AppliChem), 27.45 mM Streptomycin sulfate (AppliChem), and 2 mM glutamine (Sigma), with 2 μg/mL doxycycline (Dox) (Sigma-Aldrich), and split at around 80–90% confluency. For the recombination of libraries, 3.5 million cells were seeded out into a 10 cm plate with 10 mL DMEM without doxycycline. The next day, the cells were transfected as follows: In one tube, 7.1 μg library DNA and 0.48 μg pNLS-bxb1-recombinase were mixed with OptiMEM (Gibco) in a total volume of 710 μL. In another tube, 28.5 μL Fugene HD (Promega) was added to 685 μL OptiMEM and mixed gently. The two solutions were mixed gently by pipetting and incubated for 15 min before being added to the cells in 10 cm plates. Approximately 48 h later, 2 μg/ml doxycycline and 10 nM AP1903 (MedChemExpress) was added. The library was grown for 5 days in doxycycline before FACS profiling/sorting. A minimum of 106 cells, corresponding to approximately 150-fold coverage of the total number of variants, were maintained in the population at all times following library recombination.
5
Microbial Profiling of Cheese and Brine
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Cheese and brine samples were analyzed for viability of E. coli O157:H7, Lb. reuteri, total nonstarter LAB (NSLAB), yeasts, and molds. Five-gram samples from a cross-sectioned piece of white-brined cheese or 5 mL of brine were initially diluted with 45 mL of 0.1% peptone water and homogenized in a sterile stomacher bag for 2 min using a stomacher model 400 (Seward Ltd., London, UK). To enumerate E. coli O157:H7, 100 µL from an appropriate dilution was withdrawn and surface plated on cefixime tellurite-supplemented agar (Oxoid), overlaid with TS agar to allow recovery of injured E. coli O157:H7 cells, and incubated at 37°C for 24 h aerobically. The Lb. reuteri numbers were determined by withdrawal of 100 µL from an appropriate dilution into an empty Petri dish, and then liquefied MRS agar supplemented with penicillin G (AppliChem GmbH, Darmstadt, Germany) was poured in the Petri dishes containing the samples and mixed thoroughly before anaerobic incubation using a CO 2 generating kit (AnaeroGen, Oxoid) at 37°C for 48 h. For NSLAB, a 100-µL sample was plated on Lactobacillus-selective agar and incubated aerobically at 30°C for 5 d (Rogosa et al., 1951) (link). The numbers of yeasts and molds were enumerated by surface plating 100 µL on potato dextrose agar and incubating aerobically at 25°C for 5 d.
6
Cell Viability Assay with Chemotherapeutics
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Streptomycin and penicillin G were purchased from AppliChem (Darmstadt, Germany), while cisplatin (CP) was acquired from TEVA (Actavis D.O.O., Beograd, Serbia). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), used for the estimation of cell viability, was obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell medium (RPMI, acquired from Sigma-Aldrich) used in experiments consisted of RPMI 1640 with 20 mM N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) and L-glutamine, without sodium bicarbonate, containing 5% fetal bovine serum, 200 mg/mL Streptomycin, and 200 IU/mL penicillin [22] . All other chemicals were of analytical grade or better (Merck, Darmstadt, Germany; Sigma-Aldrich; TCI, Tokyo, Japan).
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