All CE separations were conducted on a Capillary Electrophoresis 1600 system with diode array UV detector (Agilent Technologies, G1600 A, Oregon, USA). Electrophoresis was performed in fused silica capillaries of 75 μm i.d. and of effective length of 50 cm and total 57 cm long (Agilent). Gas bubbles from all solutions and samples were removed by ultrasonic bath Sonorex (Bandelin, Berlin, G) and they were then centrifuged in a 4000 rpm speed centrifuge (Sigma, 1-6P Laboratory Centrifuge). The solutions’ pH was measured using a model pH/Ion meter-720A with an Orion 71-03 glass electrode (ThermoOrion Beverly, MA 01915-6199, USA). All of the buffers and sample solutions were filtered through a regenerated cellulose (RC) membrane filter 0.45 μm prior to analysis (La-Pha-Pack, Rockwood, TN, USA).
Sonorex
Manufactured by Bandelin
Sourced in Germany
Sonorex is a laboratory equipment product designed for ultrasonic cleaning. It utilizes high-frequency sound waves to agitate and clean various types of materials and surfaces.
Automatically generated - may contain errors
Lab products found in correlation
Sonoplus, by Bandelin (5 mentions)
Rotavapor r 114, by Büchi (2 mentions)
Whatman, by Cytiva (2 mentions)
Fused silica capillaries, by Agilent Technologies (1 mentions)
Alpha 1 2 ldplus freeze dryer, by Martin Christ (1 mentions)
Horse blood agar, by Thermo Fisher Scientific (1 mentions)
Staphaurex, by Thermo Fisher Scientific (1 mentions)
Ne 300, by New Era Pump Systems (1 mentions)
Smartlab, by Rigaku (1 mentions)
N 1 filter paper, by Cytiva (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Statistica 13.1 pl software, by StatSoft (1 mentions)
Vs 70 t, by Bandelin (1 mentions)
Alpha 1 2 ld plus, by Martin Christ (1 mentions)
Rencell cx human neural progenitor cells, by Merck Group (1 mentions)
2 propanol, by Avantor (1 mentions)
Whatman no 40 filter paper, by Cytiva (1 mentions)
Speedvac, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
40 protocols using sonorex
1
Capillary Electrophoresis for Compound Separation
2
Retrieval Analysis of Femoral Heads
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For this study, retrieved femoral heads of various designs with metallic deposition localized on the surface were selected from the retrieval archive of our hospital. Patients signed a consent statement for the safekeeping and examination of the explants. The data protection regulations were observed. The study was approved by the local Ethics Committee (registration number A 2019-0103). The selected 98 heads consisted of five different materials. The numbers of heads per material group are listed in Table 1 .
The patient data collected in reference to the selected explants were anonymized. Since many of the collected samples were not implanted at our Orthopedic Hospital, the implantation period is mostly unknown and was therefore not investigated in this study. The type of bearing materials as well as the cause for revision were considered for the evaluation of the results. After revision, all implant systems were sanitized at 95 °C. Furthermore, all femoral heads were cleaned in deionized water in an ultrasonic bath (SONOREX, BANDELIN electronic GmbH & Co., KG, Berlin, Germany) and dried with nitrogen to remove loose wear debris before examination.
An experienced and board-certified orthopedic surgeon, specially trained and approved in total joint arthroplasty at a center of excellence (Endocert-Certification Germany), was consulted for all subsequent examinations.
The patient data collected in reference to the selected explants were anonymized. Since many of the collected samples were not implanted at our Orthopedic Hospital, the implantation period is mostly unknown and was therefore not investigated in this study. The type of bearing materials as well as the cause for revision were considered for the evaluation of the results. After revision, all implant systems were sanitized at 95 °C. Furthermore, all femoral heads were cleaned in deionized water in an ultrasonic bath (SONOREX, BANDELIN electronic GmbH & Co., KG, Berlin, Germany) and dried with nitrogen to remove loose wear debris before examination.
An experienced and board-certified orthopedic surgeon, specially trained and approved in total joint arthroplasty at a center of excellence (Endocert-Certification Germany), was consulted for all subsequent examinations.
3
Phenolic Compound Extraction from Madeira Plants
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Plant material was collected in different locations of Madeira Island (Portugal) as described in Table 1. Branches were cut from healthy plants in the mentioned locations.
Leaves were lyophilized to dryness (Alpha 1-2 LD Plus freeze dryer, CHRIST), ground to powder, and stored at -20 °C. Phenolic extraction followed a previous procedure (Spínola et al., 2014) : 1 g of dry material was extracted with 25 mL of methanol in an ultrasonic bath (Bandelin Sonorex, Germany) at 35 kHz and 200 W for 60 min (room temperature). Chlorophylls (which can interfere in the analyses) were removed by adsorption on activated charcoal and extracts were filtered and concentrated to dryness in a rotary evaporator (Buchi Rotavapor R-114; USA) at 40 °C. The resulting extracts were stored at 4 °C until further analysis.
Leaves were lyophilized to dryness (Alpha 1-2 LD Plus freeze dryer, CHRIST), ground to powder, and stored at -20 °C. Phenolic extraction followed a previous procedure (Spínola et al., 2014) : 1 g of dry material was extracted with 25 mL of methanol in an ultrasonic bath (Bandelin Sonorex, Germany) at 35 kHz and 200 W for 60 min (room temperature). Chlorophylls (which can interfere in the analyses) were removed by adsorption on activated charcoal and extracts were filtered and concentrated to dryness in a rotary evaporator (Buchi Rotavapor R-114; USA) at 40 °C. The resulting extracts were stored at 4 °C until further analysis.
4
Bacterial Quantification on Implant and Tissue
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The number of bacteria associated with the implant was determined by sonicating the plate and screws in PBS for 3 min (Bandelin Sonorex at 40 kHz) and vortex mixing for 20 s before performing serial dilution and viable plate counts on 5% horse blood agar (Oxoid, Switzerland). The quantity of bacteria associated with the soft tissue and femur was determined by taking a sample of the homogenized tissue, centrifuging at 2500 g for 10 min, re-suspending in 200 μL ultrapure water and incubating for 15 min at room temperature to lyse any eukaryotic cells that may harbor intracellular bacteria. Following this incubation, 200 μL of 1.8% NaCl was added and the bacteria were re-suspended and quantified by plating serial dilutions on blood agar plates.
All agar plates were incubated for 24 h at 37 °C and any subsequent bacterial growth checked for contamination or signs of co-infection. Identification of the bacteria growing in culture-positive samples was performed for at least one colony from each culture positive animal, by a S. aureus latex agglutination assay (Staphaurex, by Remel, Thermo Scientific, Switzerland).
All agar plates were incubated for 24 h at 37 °C and any subsequent bacterial growth checked for contamination or signs of co-infection. Identification of the bacteria growing in culture-positive samples was performed for at least one colony from each culture positive animal, by a S. aureus latex agglutination assay (Staphaurex, by Remel, Thermo Scientific, Switzerland).
5
Eradication of Staphylococcus epidermidis Biofilms
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The eradication capacity of BiF2_5K7K against preformed biofilm was examined as previously described with some modifications [22 , 23 (link)]. In brief, suspensions of S. epidermidis with a final density of 106 CFU/mL were added to 96-well microplates, then covered with 96-peg lids (Nunc™ Immuno TSP Lids, Denmark). Following 24 h of incubation at 37°C, the peg lids were washed with 0.85% NaCl to remove culture media and non-adherent cells. Afterwards, the biofilms adherent to peg lids were incubated at 37°C for 24 h with different concentrations of peptide derivatives. The peg lids were subsequently washed and placed on new plates containing PBS. The biofilms on the lid were removed using a water bath sonicator (Bandelin SONOREX™, Germany). Biofilms were subsequently diluted and then placed on TSA. Minimum biofilm eradication concentrations at 50% and 90% (MBEC50 and MBEC90) refer to the concentration of the peptide at which established biofilm was eradicated by at least 50% and 90%, respectively [19 , 20 (link)].
6
Graphene Flake Dispersion and Characterization
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Graphene flake dispersions were achieved by washing of the flakes with acetone and isopropanol over filter paper and then dispersed in isopropanol by brief sonication in an ultrasonic bath (Bandelin Sonorex, 35 kHz). Then the flakes were dip-coated onto silicon substrates with a 300 nm oxide layer for Raman spectroscopy, AFM and optical microscope characterisation, and onto silicon for FTIR analysis. For XRD analysis the material was collected after washing and pasted onto a zero-background silicon holder. The transparent films were obtained by Langmuir–Blodgett technique where the graphene flakes assembled on a water surface, were transferred onto quartz glass substrate and subsequently annealed for dehydrogenation. The dehydrogenation was carried out by annealing at over 600 °C under vacuum in a tube furnace.
7
Bacterial Enumeration in Implant Infections
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Total viable bacterial counts were determined by plating bone homogenate or soft tissue homogenate on 5% horse blood agar (BA) (Oxoid). The number of bacteria associated with the implant was determined by sonicating the implant and screws in PBS for 3 min (Bandelin Sonorex at 40 kHz), vortex mixing for 10 s, and finally plating serial dilutions on BA. All BA plates were incubated at 37°C and CFU counts taken at 24 and 48 h. Mice were considered as infected when at least one sample (bone, soft tissue or implant) was culture positive. A representative colony recovered from each animal were confirmed to be S. epidermidis Epi 103.1 by Random Amplification of Polymorphic DNA (RAPD) (Versalovic et al., 1991 (link)).
8
Electrospinning of Cellulose Acetate/Nanoclay Composites
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Fibrous CA/NC composites were fabricated by electrospinning. First, CA was dissolved in a mixed-acetone/H2O solvent = 80/20 v/v at a concentration of 10 wt%. Then, three different composites were prepared by adding the respective nanoclays (10 wt% with respect to CA) to the CA spinning solution. The obtained CA/NC1, CA/NC2, and CA/NC3 mixtures were homogenized by vigorous stirring and sonication (Bandelin Sonorex, 160/640 W, 35 kHz, Berlin, Germany) for 1 h. Then, mixtures were loaded into a 10 mL Luer-Lock tip plastic syringes capped with a 20-gauge needle, placed horizontally in syringe pumps (NE-300, New Era Pump Systems, Inc., Farmingdale, NY, USA), and delivered at a constant feed rate of 3 mL/h. The electrospinning of the mixtures was conducted at an applied voltage of 25 kV, a tip-to-collector distance of 15 cm, and a collector rotation speed of 1000 rpm. Electrospun CA/NC composites were dried under vacuum at 30 °C in order to remove any solvent residues.
9
Synthesis and Deprotection of Functionalized Graphene
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First, 200 mg of GrF was dispersed in 10 mL of DMF and then subjected to ultrasonication for 10 min (Bandelin Sonoplus, type UW 3200, probe VS70T). The flask containing the dispersion was subsequently sonicated in a sonication bath for 2.5 h (Bandelin Sonorex, DT255H type, frequency 35 kHz, power 640 W, effective power 160 W). Next, 1.76 g of Boc‐Arg‐OH was added to the flask and the mixture was sonicated for a further 1 h. Afterwards, 0.44 g of potassium carbonate in 2 mL of water was added and the mixture was sonicated for a further 15 min. Subsequently, the reagents were heated under stirring at 125 °C for 48 h. The black product was separated by centrifugation and was purified by repeated centrifugal washings with DMF, water, and ethanol. The purified product (FG/Boc‐48 h_K) was then transferred to a flask to which were added 20 mL hydrochloric acid (≈5.7 m ) and around 3.75 mL acetone. The mixture was heated under stirring at 100 °C for 1 h to remove the Boc protecting group. Finally, the black product was isolated by centrifugation and was purified by successive centrifugal washings with water, acetone, and ethanol. The purified product was labeled FG/Arg‐48 h_K.
10
Functionalized Graphene Arginine Synthesis
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First, 200 mg of GrF was dispersed in 10 mL of DMF and subjected to ultrasonication for 10 min (Bandelin Sonoplus, type UW 3200, probe VS70T). The flask containing the dispersion was then sonicated in a sonication bath for 2.5 h (Bandelin Sonorex, DT255H type, frequency 35 kHz, power 640 W, effective power 160 W). Next, 1.76 g of Boc‐Arg‐OH was added to the flask and the mixture was sonicated for a further 1 h. Afterwards, the reagents were heated under stirring at 125 °C for x=24, 48, or 72 h. The black product was separated by centrifugation and was purified by repeated centrifugal washings with DMF, water, and ethanol. The purified product (FG/Boc‐xh) was transferred to a flask to which were added 20 mL hydrochloric acid (≈5.7 m ) and around 3.75 mL acetone. The mixture was heated under stirring at 100 °C for 1 h to remove the Boc protecting group. Finally, the black product was isolated by centrifugation and purified by successive centrifugal washings with water, acetone, and ethanol. The purified product was labeled FG/Arg‐xh.
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