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34 protocols using maxq 4450

1

Cultivation of Pathogenic Microbes

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Gram(+) Staphylococcus aureus (ATCC 25923) and Gram(–) Escherichia coli (ATCC 25922) were grown in 20 mL medium containing 30 g L−1 Todd-Hewitt-Bouillon (Roth) and 3 g L−1 yeast extract (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) at 37 °C overnight under constant agitation at 200 rpm on a shaking incubator (MaxQ 4450, Thermo Scientific, Marietta, OH, USA). Pathogenic yeast Candida albicans (Mya 273) was grown in 20 mL medium containing 32 g L−1 Peptone Casein (Sigma-Aldrich), 20 g L−1 yeast extract (Sigma-Aldrich), 5 g L−1 sodium chloride (VWR International, Vienna, Austria) and 5 mM sodium hydroxide (Fluka Analytical, Munich, Germany) at 37 °C overnight under constant agitation at 200 rpm on a shaking incubator (MaxQ 4450, Thermo Scientific, Marietta, OH, USA).
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2

Standardized Staphylococcus aureus Inoculation

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S. aureus was prepared for inoculation as previously published.31 (link),42 (link),61 (link) Of note, Xen36 can be isolated from contaminants due to possession of a kanamycin resistance selection marker on its lux operon. Therefore, 200 mg·mL−1 kanamycin (Sigma–Aldrich, St. Louis, MO) was added to all cultures to ensure sample purity. Xen36 was streaked onto tryptic soy agar plates (tryptic soy broth (TSB) plus 1.5% bacto agar, BD Biosciences, San Jose, CA) and cultured at 37 °C overnight. Next, single colonies of S. aureus were individually grown in TSB and again cultured overnight at 37 °C in a shaking incubator (240 r·min−1) (MaxQ 4450, ThermoFisher, Grand Island, NY). After a 2-h subculture of a 1:50 dilution of the resultant culture, mid-logarithmic phase bacteria were attained. Finally, using a centrifuge, bacterial cells were pelleted, re-suspended, and washed in phosphate-buffered saline (PBS). Bacterial inoculums (5 × 101, 1 × 102, 1 × 103, and 1 × 104 CFUs in 2 μL PBS) were approximated by measuring the absorbance at 600 nm [A600, Biomate 3 (Thermo)].
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3

Preparation and Characterization of Aib2-Oxm Fibrils

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The preparation of Aib2-Oxm solutions was carried out in a class 2 cabinet using sterile glass vials. Aib2-Oxm was dissolved at 10-20 mg mL-1 in sterile NaOH (4.3 mM) and 0.09% saline solution (diluted from the 0.9% saline solution) to achieve a pH between 7.0 and 7.3. The solution was passed through a 0.22 µm pore size membrane, and peptide concentration was measured before diluting the solution to 10 mg mL-1 using 0.09% saline. The solution was then incubated at 37°C for 3 to 6 days with orbital shaking (200 rpm, ThermoScientific MaxQ 4450 benchtop orbital shaker) and then 2–3 days without agitation. Thereafter, 1 mg mL-1 of fibrillar Aib2-Oxm was incubated with 10 mg mL-1 free peptide in 0.09% for 1 week at RT without agitation. Finally, the 10 mg mL-1 fibrillar Oxm solution was diluted to varying concentration between 1 to 4 mg mL-1 using sterile 0.09% saline, incubated at RT for 2–9 days before being stored at 4°C. The conversion yield was assessed by measuring the concentration of the remaining free peptide. The fibrillar material was separated from the free peptide in solution after centrifugation of an aliquot of the solution for 30 min at 16 200 x g and filtering the supernatant through a 50 kDa molecular weight cut-off membrane.
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4

Retinoic Acid Sorption Assay

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In this assay, excess amounts of RA and the systems were placed in glass vials, and 5 mL of distilled water was added. The samples were incubated for 24 h at 298.15 K at a constant speed of 75 rotations per minute (rpm) using a laboratory incubator MaxQ 4450 (Thermo Scientific, Waltham, MA, USA). Subsequently, the suspensions were filtered through 0.22 μm and analyzed by the HPLC method. All measurements were performed in triplicate.
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5

Solubility Studies of Crystalline GEN

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The solubility studies were carried out in distilled water. A total of 6 mg of crystalline GEN and a quantity of co-amorphous systems samples in excess of its expected saturated solubility were administered with 5 mL of a medium and placed in laboratory incubator MaxQ 4450 (Thermo Scientific, Waltham, MA, USA) maintained at 25 °C, agitation speed of 75 rpm and protected from light. After 2 h, the obtained solutions were filtered through a 0.45-μm nylon membrane syringe filter and injected into the HPLC system for GEN content analysis.
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6

Comprehensive Yeast Strain Cultivation Protocol

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All the strains used in this study are listed in Table S1 (Appendix 1), which includes information about the origin (source), place of primary isolation, and actual taxonomic position for each tested strain. Strains were routinely cultured on YPD (Yeast extract Peptone Dextrose) semi-solid medium, followed by liquid medium: 1% yeast extract, 2% bacto-peptone and in case of semi-solid media 2% bacto-agar (BD Difco, Sparks, MD, USA, cat. no. REF212720, REF211820, and REF212720) and filter-sterilized 2% glucose (VWR International LLC, West Chester, PA, USA, cat. no. BDH0230). In case of Titan induction experiment, cells were grown overnight in 5 ml YNB (Yeast Nitrogen Base) liquid medium: 0.67% Yeast Nitrogen Base with amino acids, pH 5.5 (Sigma cat. no. Y1250, St. Louis, MO, USA), 2% glucose at 30°C with horizontal shaking at 220 rpm (Thermo Scientific, MAXQ4450).
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7

Evaluating S. baicalensis Radix Antioxidant and Anti-Inflammatory Potential

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Systems extract of S. baicalensis radix with chitosan were tested to evaluate their biological activity in periodontal diseases. The antioxidant potential (ABTS assay and ferrous ion chelating activity), anti-inflammatory effect (anti-hyaluronidase activity), and microbiological activity of binary mixtures compared with the S. baicalensis radix extract alone were estimated.
The solutions to antioxidant and anti-hyaluronidase activity studies of S. baicalensis radix lyophilized extract and binary mixtures were prepared by shaking (400 rpm min−1) with buffer solutions at pH 6.6 on a shaker (Thermo Scientific MaxQ 4450, Waltham, MA, USA) for 30 min. at 37 °C and then centrifuging at 4100 rpm min−1 for 20 min (Nüve NF 800, Ankara, Turkey) to produce a clear supernatant. The IC50 values were calculated with OriginPro 9 software. All experiments were performed six times.
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8

Dissolution kinetics of NAR compounds

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The dissolution test was performed at 298.15 K. Excess amounts of NAR and analogical excess amounts of NAR in the systems were added to 4 mL of distilled water and agitated for 24 h at a constant speed of 75 rotations per minute (rpm) using a laboratory incubator MaxQ 4450 (Thermo Scientific, Waltham, MA, USA). Then, the suspensions were filtered through a 0.22 μm filter and analyzed by the HPLC method. All measurements were performed in triplicate.
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9

Xen36 S. aureus Kanamycin Growth

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S. aureus Xen 36 possesses a kanamycin resistance marker on the lux operon. Bacteria were streaked onto tryptic soy agar plates (tryptic soy broth [TSB] plus 1.5% Bacto agar [BD Biosciences, Franklin Lakes, NJ]) containing 200ug/ml kanamycin (Sigma-Aldrich) and grown overnight at 37°C to select for Xen36 as previously described.[26 (link)] Single colonies of Xen36 were cultured in TSB and grown overnight at 37°C in a shaking incubator (240 rpm) (MaxQ 4450; Thermo, Waltham, MA). Mid-logarithmic phase bacteria were obtained after a 2-hour subculture of a 1:50 dilution of the overnight culture. Bacterial cells were pelleted, resuspended and washed 3 times in phosphate buffered saline (PBS). Bacterial inocula (1x103 CFU/ml) were estimated by measuring the absorbance at 600 nm (Biomate 3; Thermo).
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10

Evaluating Ho-166 Graphene Oxide Nanoparticle Release

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Two hundred μL of 166Ho-GONs or 166Ho-GONs-PEG (0.5 mg of GONs/mL) were placed in a dialysis cup (Slide-A-Lyzer MINI dialysis®, 3500 MWCO) which was subsequently dialyzed against 20 mL of phosphate buffered saline (PBS) at 37 °C and shaken at 100 rpm (Thermo MAXQ 4450). A sample of the dialysate was drawn at pre-determined time points (1, 3, 6, 12, 24, 36 and 48 h) and the leached amount of 166Ho was quantified with a γ-counter.
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