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1.
J Chromatogr A ; 1635: 461752, 2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33333350

RESUMO

Extracellular vesicles (EVs) are membrane enclosed vesicles (<1 µm), such as exosomes (30-150 nm), involved in cell communication, which have important biological implications. In this study, EV preparations were enriched for exosomes from human serum by polyethylene glycol (PEG) precipitation. Different variables of the PEG precipitation method (i.e. concentration of PEG, filtration and centrifugation of the resuspended pellets) were evaluated by measuring the size of the isolated particles by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). In addition, a novel capillary electrophoresis-ultraviolet diode array (CE-UV-DAD) method was developed to obtain characteristic multiwavelength electrophoretic profiles of the EV preparations. Using EV preparations precipitated with 10% m/v of PEG, a background electrolyte (BGE) of 0.1 M Tris and 0.25 M boric acid at pH 7.9 with 0.5% m/v of hydroxypropyl cellulose (HPC) allowed reducing the adsorption of the EVs to the inner wall of the fused silica separation capillary. Sodium dodecyl sulfate (SDS) at 0.1% m/v was also necessary to enhance dispersibility, while homogenizing the charge of the particles to improve the size-dependent separation induced by HPC. Under these optimized conditions, a characteristic electrophoretic multiwavelength profile of the EV preparation and a standard of exosomes was obtained, and separation showed excellent reproducibility and appropriate analysis times. The obtained electrophoretic fingerprints are a simple, effective and complementary tool for the quality control of EV preparations.


Assuntos
Técnicas de Química Analítica/métodos , Eletroforese Capilar , Exossomos , Vesículas Extracelulares , Soro/química , Centrifugação , Técnicas de Química Analítica/instrumentação , Eletroforese Capilar/instrumentação , Filtração , Humanos , Nanopartículas , Reprodutibilidade dos Testes
2.
J Environ Manage ; 280: 111745, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33298394

RESUMO

Centrifuge are common machines used for sludge thickening and dewatering in wastewater treatment plant but performances and optimal setting parameters are often difficult to predict and optimize, owing to complex mechanisms occurring during separation and high shear stresses undergone by flocs. Laboratory tests are necessary to assess performances and to screen chemicals and dosage in order to decrease optimization time at full scale. They include volatile matters characterization, drainage and CST tests vs mixing time, basket spin tests with shearing, limit dryness determination. The ability of these tests, to assess sludge dehydration and/or to select polymer and dosage, is discussed and is compared with performances obtained in a screw centrifuge decanter at lab and full scale.


Assuntos
Esgotos , Eliminação de Resíduos Líquidos , Parafusos Ósseos , Centrifugação , Polímeros , Água
3.
Plast Reconstr Surg ; 146(6): 749e-758e, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33234959

RESUMO

BACKGROUND: Lipofilling is one of the most often performed surgical procedures in plastic and reconstructive surgery. Lipoaspirates provide a ready source of stem cells and secreted factors that contribute to neoangiogenesis and fat graft survival. However, the regulations about the enrichment of these beneficial cells and factors are ambiguous. In this study, the authors tested whether a combination of centrifugation and homogenization allowed the enrichment of viable stem cells in lipoaspirates through the selective removal of tumescent solution, blood, and released lipids without significantly affecting the cell secretome. METHODS: Human lipoaspirate was harvested from six different patients using water jet-assisted liposuction. Lipoaspirate was homogenized by first centrifugation (3584 rpm for 2 minutes), shear strain (10 times intersyringe processing), and second centrifugation (3584 rpm for 2 minutes). Stem cell enrichment was shown by cell counting after stem cell isolation. Lipoaspirate from different processing steps (unprocessed, after first centrifugation, after homogenization, after second centrifugation) was incubated in serum-free cell culture medium for mass spectrometric analysis of secreted proteins. RESULTS: Lipoaspirate homogenization leads to a significant 2.6 ± 1.75-fold enrichment attributable to volume reduction without reducing the viability of the stem cells. Protein composition of the secretome did not change significantly after tissue homogenization. Considering the enrichment effects, there were no significant differences in the protein concentration of the 83 proteins found in all processing steps. CONCLUSIONS: Stem cells can be enriched mechanically without significantly affecting the composition of secreted proteins. Shear-assisted enrichment of lipoaspirate constitutes no substantial manipulation of the cells' secretome.


Assuntos
Tecido Adiposo/citologia , Proteoma/metabolismo , Transplante de Células-Tronco , Células-Tronco/metabolismo , Adulto , Idoso , Contorno Corporal/métodos , Contagem de Células , Separação Celular/métodos , Centrifugação/efeitos adversos , Meios de Cultura Livres de Soro , Feminino , Sobrevivência de Enxerto/fisiologia , Voluntários Saudáveis , Humanos , Lipectomia/métodos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Neovascularização Fisiológica/fisiologia , Cultura Primária de Células/métodos , Proteoma/análise , Proteômica , Resistência ao Cisalhamento
4.
Plast Reconstr Surg ; 146(6): 1275-1284, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33234957

RESUMO

BACKGROUND: The stromal vascular fraction can improve volume retention after fat grafting, but the optimal stromal vascular fraction extraction method remains controversial. This study investigated the effect of mechanical vibration on stromal vascular fraction activity and explored the efficacy of vibration as a new extraction method compared to centrifugation, enzyme digestion, and nanoemulsion methods. METHODS: Twenty-four rabbits were divided into three groups, and adipose tissue was harvested from the scapular region of each rabbit. In the first group, stromal vascular fraction was extracted from adipose tissue by vibration with different frequencies and durations. Cell counts and colony formation were assessed to determine the optimal vibration parameters. In the second group, stromal vascular fraction was extracted by the four methods, and the cell counts, proliferation, and adipogenic capabilities were observed in vitro. In the third group, adipose tissue mixed with stromal vascular fraction extracted by means of the four methods was grafted into rabbit ears. Volume retention and histologic changes were evaluated over 24 weeks. RESULTS: Stromal vascular fraction activity was not influenced by low-frequency (≤45 Hz) and short-duration (≤20 minutes) vibrations. Vibration at 30 Hz for 15 minutes was most efficient for stromal vascular fraction extraction. In vitro, stromal vascular fraction extracted by vibration showed advantages for cell viability. In vivo, the vibration group showed a more normal tissue morphology and a higher retention rate (60.68 ± 7.07 percent) than the enzyme digestion (31.88 ± 4.99 percent), centrifugation (43.76 ± 4.32 percent), and nanoemulsion groups (21.79 ± 3.57 percent) (p < 0.05). CONCLUSION: Vibration at 30 Hz for 15 minutes is recommended as a novel nonenzymatic method to extract stromal vascular fraction with high activity.


Assuntos
Tecido Adiposo/transplante , Células Estromais/transplante , Coleta de Tecidos e Órgãos/métodos , Vibração , Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/citologia , Tecido Adiposo/diagnóstico por imagem , Animais , Separação Celular/métodos , Centrifugação , Masculino , Modelos Animais , Coelhos , Transplante Autólogo/métodos , Ultrassonografia
5.
Plast Reconstr Surg ; 146(6): 1285-1293, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33234958

RESUMO

BACKGROUND: Regenerative cell strategies rely on stromal cell implants to attain an observable clinical outcome. However, the effective cell dose to ensure a therapeutic response remains unknown. To achieve a higher cell dose, the authors hypothesized that reducing the volume occupied by mature adipocytes in lipoaspirate will concentrate the stromal vascular fraction present in the original tissue. METHODS: Human standardized lipoaspirate (n = 6) was centrifuged (1200 g for 3 minutes) and the water phase was discarded. Mechanical disaggregation was achieved by shearing tissue through 2.4- and 1.2-mm Luer-to-Luer transfers. After a second centrifugation (800 g for 10 minutes), stromal cell aggregates were separated from the supernatant oil phase. Lipoaspirate percentage composition was determined by its constituent weights. Cell content was measured by total DNA quantification, and partial cell viability was determined by image cytometry. Tissue sections were evaluated histologically (hematoxylin and eosin and Masson trichrome stains). RESULTS: Stromal cell aggregates reduced the standardized lipoaspirate mass to 28.6 ± 4.2 percent. Accordingly, the cell density increased by 222.6 ± 63.3 percent (from 9.9 ± 1.4 million cells/g to 31.3 ± 6.6 million cells/g; p < 0.05). Cell viability was unaffected in stromal cell aggregates (71.3 ± 2.5 percent) compared to standardized lipoaspirate (72.2 ± 2.3 percent), and histologic analysis revealed high-density areas enriched with stromal cells (622.9 ± 145.6 percent) and extracellular matrix (871.2 ± 80.3 percent). CONCLUSION: Stromal cell aggregates represent a biological agent that triplicates the cell density versus unprocessed lipoaspirate, low on oil and water fluids, and enriched extracellular matrix components.


Assuntos
Tecido Adiposo/transplante , Células Estromais/transplante , Coleta de Tecidos e Órgãos/métodos , Adipócitos/fisiologia , Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/citologia , Adolescente , Adulto , Contagem de Células/métodos , Separação Celular/métodos , Sobrevivência Celular , Centrifugação , DNA/isolamento & purificação , Voluntários Saudáveis , Humanos , Lipectomia , Pessoa de Meia-Idade , Células Estromais/fisiologia , Transplante Autólogo/métodos , Adulto Jovem
6.
PLoS One ; 15(10): e0240134, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33027285

RESUMO

Blood Concentrates (BCs) are autologous non-transfusional therapeutical preparations with biological properties applied in tissue regeneration. These BCs differ in the preparation method, in fibrin network architecture, growth factors release as well as in platelet/cell content. Methodological changes result in distinct matrices that can compromise their clinical effectiveness. The present study evaluated the influence of different g-forces and types of tubes in the release of vascular endothelial growth factor (VEGF) from platelet-rich fibrin (PRF) as a function of time. The PRF-like samples were obtained with three g-forces (200, 400, and 800 x g) for 10 minutes in pure glass tubes or in polystyrene-clot activator tubes. Scanning and Transmission electron microscopy was used to morphometric analyzes of PRF's specimens and flow cytometry was used to quantify VEGF slow release until 7 days. Our results showed that platelets were intact and adhered to the fibrin network, emitting pseudopods and in degranulation. The fibrin network was rough and twisted with exosomic granulations impregnated on its surface. An increase in the concentration of VEGF in the PRF supernatant was observed until 7 days for all g forces (200, 400 or 800 xg), with the highest concentrations observed with 200 x g, in both tubes, glass or plastic. Morphological analyzes showed a reduction in the diameter of the PRF fibers after 7 days. Our results showed that g-force interferes with the shape of the fibrin network in the PRF, as well as affect the release of VEGF stored into platelets. This finding may be useful in applying PRF to skin lesions, in which the rapid release of growth factors can favor the tissue repair process. Our observations point to a greater clarification on the methodological variations related to obtaining PRF matrices, as they can generate products with different characteristics and degrees of effectiveness in specific applications.


Assuntos
Plaquetas/metabolismo , Fibrinólise , Fibrina Rica em Plaquetas/metabolismo , Engenharia Tecidual/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Plaquetas/ultraestrutura , Centrifugação/efeitos adversos , Centrifugação/métodos , Feminino , Fibrina/metabolismo , Fibrina/ultraestrutura , Voluntários Saudáveis , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Fator A de Crescimento do Endotélio Vascular/análise
7.
AAPS PharmSciTech ; 21(7): 265, 2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-33006045

RESUMO

This study used dual asymmetric centrifugation (DAC) to produce a topical vehicle for Pirfenidone (Pf; 5-methyl-1-phenyl-2[1H]-pyridone)-a Food and Drug Administration-approved antifibrotic drug indicated for idiopathic fibrosis treatment. Pf was loaded (8 wt%) in a poloxamer nanoemulsion gel (PNG) formulation consisting of water (47.8 wt%), triacetin (27.6 wt%), poloxamer 407 (P407, 13.8 wt%), polysorbate 80 (1.8 wt%), and benzyl alcohol (0.9 wt%). To our knowledge, poloxamer gels are typically processed with either high-shear methods or temperature regulation and have not been emulsified using DAC. Using a single-step emulsification process, 2 min mixed at 2500 RPM resulted in the lowest Pf loading variability with a relative standard deviation (RSD) of 0.96% for a 1.5 g batch size. Batch sizes of 15 g and 100 g yield higher RSD of 4.18% and 3.05%, respectively, but still in compliance with USP guidelines. Ex vivo permeation in full thickness porcine skin after 24 h showed total Pf permeation of 404.90 ± 67.07 µg/cm2. Tested in vitro on human dermal fibroblasts stimulated with transforming growth factor-beta 1 (TGF-ß1), Pf-PNG resulted in a > 2 fold decrease in α-SMA expression over vehicle control demonstrating that formulated Pf retained its biological activity. One-month stability testing at 25°C/60% relative humidity (RH) and 40°C/75% RH showed that % drug content, release kinetics, and biological activity were largely unchanged for both conditions; however, pH decreased from 6.7 to 5.5 (25°C/60% RH) and 4.5 (40°C/75% RH) after 1 month. Overall, these data demonstrate the utility of DAC to rapidly and reproducibly prepare lab-scale batches of emulsified gels for pharmaceutical formulation development.


Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Poloxâmero/química , Piridonas/administração & dosagem , Administração Tópica , Animais , Centrifugação , Química Farmacêutica/métodos , Emulsões/metabolismo , Excipientes/química , Géis/química , Humanos , Absorção Cutânea , Suínos , Temperatura
8.
Int J Hematol ; 112(5): 614-620, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32929688

RESUMO

Accurate clotting time assay results are vital, as the test is employed to indicate the amount of oral anticoagulant to be prescribed, while it is also used for screening the hemorrhagic and thrombotic diseases. The procedure chosen for preparation of a patient blood sample including centrifugation can contribute to significant differences in the results obtained. Thus, for the purpose of proposing a standardized method to appropriately prepare blood samples prior to assay, the Japanese Society of Laboratory Hematology organized the Working Group for Standardization of Sample Preparation for Clotting Time Assays (WG). Following reviews of previously announced guidelines and original experimental results, consensus was obtained by the WG, with the main findings as follows. (1) The recommended anticoagulant in the blood collection tube is sodium citrate solution at 0.105-0.109 M (3.13-3.2%). (2) Whole blood samples should be stored at room temperature (18-25 ˚C) within 1 h of collection from the patient. (3) For plasma preparation, centrifugation at 1500 × g should be performed for at least 15 min or at 2000 × g for at least 10 min at room temperature. (4) After the plasma sample is prepared, it should be stored at room temperature and assayed within 4 h.


Assuntos
Testes de Coagulação Sanguínea/métodos , Testes de Coagulação Sanguínea/normas , Coleta de Amostras Sanguíneas/métodos , Coleta de Amostras Sanguíneas/normas , Consenso , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Centrifugação , Humanos
9.
J Vis Exp ; (162)2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32925882

RESUMO

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field. Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.


Assuntos
Neoplasias Encefálicas/patologia , Núcleo Celular/metabolismo , Cromatina/metabolismo , Congelamento , Sequenciamento de Nucleotídeos em Larga Escala , RNA-Seq , Transposases/metabolismo , Centrifugação , Dissecação , Citometria de Fluxo , Glioma/patologia , Humanos , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Transposases/genética
10.
Environ Monit Assess ; 192(10): 662, 2020 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-32979107

RESUMO

The centrifuge-less dispersive liquid-liquid microextraction (DLLME) technique was used to separate selenium species in aqueous samples. According to the salting-out effect, a simple approach was used to eliminate the centrifugation step. The optimization of the independent variables was performed using chemometric methods. Under optimal conditions, this methodology was statistically validated. The linearity was between 20 and 300 µg L-1. The limit of detection and quantification were calculated 3.4 µg L-1 and 10.4 µg L-1, respectively. The values of reproducibility and repeatability were determined ≤ 9.5% and ≤ 6.4, respectively. The possibility of the method was successfully assessed by analyzing the analytes in real samples clarified satisfactory recoveries (98.1-101.4% for Se (IV) and 98.4-101.5% for Se (VI)).


Assuntos
Microextração em Fase Líquida , Selênio/análise , Poluentes Químicos da Água/análise , Centrifugação , Monitoramento Ambiental , Limite de Detecção , Reprodutibilidade dos Testes
11.
PLoS One ; 15(9): e0239228, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946482

RESUMO

Artificial gravity through short-arm centrifugation has potential as a multi-system countermeasure for deconditioning and cranial fluid shifts that may underlie ocular issues in microgravity. However, the optimal short-arm centrifugation protocol that is effective whilst remaining tolerable has yet to be determined. Given that exposure to centrifugation is associated with presyncope and syncope and in addition motion sickness an intermittent protocol has been suggested to be more tolerable. Therefore, we assessed cardiovascular loading and subjective tolerability of daily short arm centrifugation with either an intermittent or a continuous protocol during long-term head-down bed rest as model for microgravity exposure in a mixed sex cohort. During the Artificial Gravity Bed Rest with European Space Agency (AGBRESA) 60 day 6° head down tilt bed rest study we compared the tolerability of daily +1 Gz exposure at the center of mass centrifugation, either performed continuously for 30 minutes, or intermittedly (6 x 5 minutes). Heart rate and blood pressure were assessed daily during centrifugation along with post motion sickness scoring and rate of perceived exertion. During bed rest, 16 subjects (6 women, 10 men), underwent 960 centrifuge runs in total. Ten centrifuge runs had to be terminated prematurely, 8 continuous runs and 2 intermittent runs, mostly due to pre-syncopal symptoms and not motion sickness. All subjects were, however, able to resume centrifuge training on subsequent days. We conclude that both continuous and intermittent short-arm centrifugation protocols providing artificial gravity equivalent to +1 Gz at the center of mass is tolerable in terms of cardiovascular loading and motion sickness during long-term head down tilt bed rest. However, intermittent centrifugation appears marginally better tolerated, albeit differences appear minor.


Assuntos
Centrifugação , Gravidade Alterada/efeitos adversos , Enjoo devido ao Movimento , Repouso em Cama , Pressão Sanguínea , Estudos de Coortes , Feminino , Decúbito Inclinado com Rebaixamento da Cabeça , Voluntários Saudáveis , Frequência Cardíaca , Humanos , Masculino
12.
J Dairy Sci ; 103(10): 8782-8790, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32828509

RESUMO

The objective of this work was to determine the effect of milk bactofugation on the counts and microbial diversity of mesophilic (MT), psychrotrophic (PT), and thermophilic (TT) thermoduric bacteria and its potential as a technological method to remove spoilage microorganisms resistant to pasteurization. Different batches of raw milk from 69 dairy farms divided into sets in 3 bulk tanks (A, B, C) were evaluated at different times during the technological process. As the raw milk was preheated (∼55°C) immediately before bactofugation (10,000 × g), the effect of bactofugation was estimated by comparing the counts in raw, preheated, and bactofuged milk. This centrifugation was sufficient to reduce the isolation of 88% of the MT in preheated milk. For PT, it was possible to verify a reduction of 72.5% in batch C. The TT were not recovered at higher detection limits (<5 cfu/mL). For diversity, 310 isolates were identified using a molecular approach; 15 species of contaminating thermoduric bacteria were identified from raw and preheated milk, and only 6 species were recovered in bactofuged milk. Only MT were recovered from the bactofuged milk, mainly the species Lysinibacillus fusiformis (61.7%) and Bacillus licheniformis (12.3%). Both species are known to be endospore-forming psychrotrophs and have proteolytic or lipolytic activity. The bactofugation of raw milk reduced the number of isolates of B. licheniformis, Bacillus toyonensis, Micrococcus aloeverae, and Aestuariimicrobium kwangyangense by 33, 43, 86, and 92%, respectively, and reduced the isolates of Macrococcus caseolyticus, Lysinibacillus varians, Carnobacterium divergens, Microbacterium hominis, Kocuria indica, Micrococcus yunnanensis, Gordonia paraffinivorans, Bacillus invictae, and Kocuria kristinae to undetectable levels. The results of this study indicate that bactofugation can be applied by the dairy industry to reduce pasteurization-resistant microorganisms in combination with prophylactic measures to prevent the contamination of raw milk by spores and vegetative forms of bacteria.


Assuntos
Bactérias Termodúricas/isolamento & purificação , Centrifugação/métodos , Leite/microbiologia , Actinobacteria/isolamento & purificação , Animais , Bacillaceae/isolamento & purificação , Bacillus/isolamento & purificação , Bactérias Termodúricas/classificação , Carnobacterium/isolamento & purificação , Micrococcaceae/isolamento & purificação , Micrococcus/isolamento & purificação , Propionibacteriaceae/isolamento & purificação , Staphylococcaceae/isolamento & purificação
13.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(8): 1025-1030, 2020 Aug 15.
Artigo em Chinês | MEDLINE | ID: mdl-32794673

RESUMO

Objective: To identify a more popularized preparation protocol of leukocytes-rich platelet-rich plasma (L-PRP) for higher tolerance rate. Methods: The peripheral blood samples of 76 volunteers (45.0 mL/case) were mixed with 5 mL sodium citrate injection for blood transfusion, and L-PRP was prepared by twice centrifugations. All blood samples were divided into three groups according to the parameters of twice centrifugation: experimental group A (12 cases, 400× g, 10 minutes for the first time and 1 100× g, 10 minutes for the second time), experimental group B (27 cases, 800× g, 10 minutes for the first time and 1 100× g, 10 minutes for the second time), and control group (37 cases, 1 360× g, 10 minutes for the first time and 1 360× g, 10 minutes for the second time). The platelet recovery rate and platelet and leukocyte enrichment coefficient of L-PRP in each group were calculated and compared. Results: After removal of abnormal blood samples (platelet recovery rate was more than 100% or white thrombus), the remaining 55 cases were included in the statistical analysis, including 10 cases in experimental group A, 21 cases in experimental group B, and 24 cases in control group. The platelet enrichment coefficient and platelet recovery rate of experimental group B were significantly higher than those of experimental group A and control group ( P<0.05); there was no significant difference between experimental group A and control group ( P>0.05). There was no significant difference in leukocyte enrichment coefficient between experimental groups A, B, and control group ( P>0.05). Conclusion: The preparation quality of PRP is affected by various factors, including centrifugal force, centrifugal time, temperature, and operation process, etc. Twice centrifugation (800× g, 10 minutes for the first time and 1 100× g, 10 minutes for the second time) is an ideal and feasible centrifugation scheme, which can obtain satisfactory platelet recovery rate and enrichment coefficient with thicker buffy coat, which can reduce the fine operation requirements for operators, improve the fault tolerance rate and generalization.


Assuntos
Plasma Rico em Plaquetas , Plaquetas , Centrifugação , Humanos , Leucócitos
14.
Bioresour Technol ; 316: 123866, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32745999

RESUMO

Centrifugation is very common in the production and treatment of lignocellulose for applications like pretreatment for enzymatic hydrolysis, but it is not certain whether it affects applications of lignocellulose and almost no one realizes this problem. This study investigated the effects of centrifugation on the characteristics and enzymatic hydrolysis of poplar fibers with high lignin content. The results showed that centrifugation inhibited the enzymatic hydrolysis of fiber, but fiber characteristics and enzymatic digestibility fluctuated with increasing centrifugation time. Centrifugation for about 15 min had the least effect on fiber properties while centrifugation for 30 min had the least effect on enzymatic hydrolysis. The water retention value was closely related to the enzymatic digestibility, but the pore characteristics and crystallinity index could not reflect the enzymatic accessibility of the fiber. This article will provide useful references for the enzymatic hydrolysis of lignocellulose and other high-value applications to improve production efficiency furtherly.


Assuntos
Celulose , Lignina , Centrifugação , Hidrólise , Xilanos
15.
J Chromatogr A ; 1627: 461398, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823103

RESUMO

A new mode of dispersive solid phase extraction based on in situ formation of adsorbent in aqueous phase has been introduced as an efficient method for the extraction of some pesticide residues in fruit juice samples. In this method, polycarbonate which is an inexpensive polymer is used as an adsorbent for the first time. The method is followed by dispersive liquid-liquid microextraction for more enrichment of the analytes. In the present study, a proper amount of the polymer is dissolved in N,N-dimethyl formamide and the obtained solution is injected into an aqueous phase containing the analytes. After injection, polycarbonate particles are formed and adsorbed the analytes. Then, the adsorbent is separated from the aqueous solution and eluted by acetone. The obtained acetone phase is mixed with 1,1,1-trichloroethane and the mixture is dispersed into deionized water and a cloudy solution is formed. Ultimately, after centrifugation, the obtained sedimented phase containing the extracted analytes is injected into gas chromatography-flame ionization detection. In the proposed method, the adsorbent synthesis step, which often is a time-consuming, expensive, and laborious step in most adsorbent-based sample preparation methods, has been removed. Moreover, there is no need for sonication or vortex agitation. Under the optimized experimental conditions, the relative standard deviation was equal or less than 7% for intra- (n = 6) and inter-day (n = 5) precisions at a concentration of 50 µg L-1 of each pesticide. The limits of detection and quantification were in the ranges of 0.34-1.2 and 1.1-4.0 µg L-1, respectively. In addition, extraction recoveries and enrichment factors varied in the ranges of 44-89% and 220-443, respectively.


Assuntos
Sucos de Frutas e Vegetais/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Resíduos de Praguicidas/análise , Centrifugação , Limite de Detecção , Microextração em Fase Líquida/métodos , Resíduos de Praguicidas/isolamento & purificação , Extração em Fase Sólida , Solventes/química , Triazinas/análise , Triazinas/isolamento & purificação , Água/química
16.
PLoS One ; 15(8): e0237930, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841274

RESUMO

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.


Assuntos
Microssomos/metabolismo , Proteômica , Animais , Células CHO , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centrifugação , Cricetinae , Cricetulus , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Ontologia Genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Microssomos/ultraestrutura , Mitocôndrias/ultraestrutura , Proteoma/metabolismo , Software , Frações Subcelulares/metabolismo
17.
Exp Parasitol ; 217: 107965, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32818513

RESUMO

Saturated salt floatation method is widely used for coccidian oocyst purification. However, the repeated procedures and inefficient oocysts recovery rate are a continuous challenge. This study aimed to investigate the best suitable floatation solution, along with optimal centrifugation speed and time for Eimeria tenella (E. tenella) oocyst and sporocyst purification. Different floatation solutions i-e, saturated salt, Sheather's sugar and sodium hypochlorite (NaClO) at 20-60% concentrations were used to purify oocyst. It was found that about 96.99% oocysts (8609×g for 10 min) were recovered under these conditions without any effect on the viability of sporocysts. The recovery rate of oocysts using 50% NaClO (V/V) was significantly higher than 35% saturated salt flotation solution (P < 0.05). The optimal method for purification of oocysts based our experimentation was centrifugation at 8609×g for 3 min using 50% NaClO floatation solution, and the optimized centrifugation conditions for improved recovery of sporocysts (about 99.3%) were at 2152×g for 5 min. The present study provided a better method for the coccidian oocyst purification, which could be successfully adopted as a better alternative to existing techniques commonly used for investigations/research pertaining to coccidia.


Assuntos
Centrifugação/normas , Eimeria tenella/isolamento & purificação , Análise de Variância , Animais , Galinhas , Eimeria tenella/crescimento & desenvolvimento , Fezes/parasitologia , Oocistos/isolamento & purificação , Oxidantes/administração & dosagem , Distribuição Aleatória , Hipoclorito de Sódio/administração & dosagem , Organismos Livres de Patógenos Específicos , Fatores de Tempo
18.
J Chromatogr A ; 1625: 461340, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709363

RESUMO

In this study, a magnetized polyethylene composite has been prepared using ball milling procedure and employed as an efficient sorbent in magnetic dispersive solid phase extraction combined with dispersive liquid-liquid microextraction. This method has been utilized for the extraction and preconcentration of some pesticides from fruit juices prior to their quantification by gas chromatography-flame ionization detection. The prepared sorbent consisted of the natural iron oxide (obtained from sand) coated with polyethylene. In the present work, first a few mg of the magnetic composite is added into an aqueous solution containing the analytes and vortexed. After that the analytes are eluted with iso-propanol from the surface of the composite particles separated in the presence of a strong external magnetic field. For further enrichment of the analytes, 1,2-dibromoethane (at µL-level) as an extraction solvent is mixed with the obtained eluent and hastily injected into deionized water. The composite was characterized using techniques including vibrating sample magnetometry, X-ray diffraction, scanning electron microscopy, energy-dispersive X-ray spectroscopy, Brunauer-Emmett-Teller nitrogen sorption, and Fourier transform infrared spectrophotometry. Under optimal conditions, the method provided low limits of detection (0.94-1.9 µg L-1) and quantification (3.2-5.9 µg L-1), high enrichment factors (570-692), good linearity (r2 ≥ 0.994), and satisfactory repeatabilities (relative standard deviations ≤ 8% for intra- and inter-day precisions at a concentration of 15 µg L-1 of each analyte).


Assuntos
Fenômenos Magnéticos , Praguicidas/isolamento & purificação , Polietileno/química , Polietileno/síntese química , Adsorção , Centrifugação , Cromatografia Gasosa , Sucos de Frutas e Vegetais/análise , Concentração de Íons de Hidrogênio , Limite de Detecção , Microextração em Fase Líquida , Concentração Osmolar , Praguicidas/análise , Praguicidas/química , Reprodutibilidade dos Testes , Extração em Fase Sólida , Solventes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Fatores de Tempo , Difração de Raios X
19.
PLoS One ; 15(7): e0235793, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32634162

RESUMO

Extracellular vesicles (EVs) are small vesicles secreted from cells. They have crucial biological functions in intercellular communications and may even be biomarkers for cancer. The various methods used to isolate EVs from body fluid and cell culture supernatant have been compared in prior studies, which determined that the component yield and physical properties of isolated EVs depend largely on the isolation method used. Several novel and combined methods have been recently developed, which have not yet been compared to the established methods. Therefore, the purpose of this study is to compare the physical and functional differences in EVs isolated using a differential centrifugation method, the precipitation-based Invitrogen kit, the ExoLutE kit, and the Exodisc, of which the latter two were recently developed. We investigated the properties of EVs isolated from non-infected and Kaposi's sarcoma-associated herpesvirus-infected human umbilical vein endothelial cells using each method and determined the yields of DNA, RNA, and proteins using quantitative polymerase chain reaction and bicinchoninic acid assays. Additionally, we determined whether the biological activity of EVs correlated with the quantity or physical properties of the EVs isolated using different methods. We found that Exodisc was the most suitable method for obtaining large quantities of EVs, which might be useful for biomarker investigations, and that the EVs separated using Exodisc exhibited the highest complement activation activity. However, we also found that the functional properties of EVs were best maintained when differential centrifugation was used. Effective isolation is necessary to study EVs as tools for diagnosing cancer and our findings may have relevant implications in the field of oncology by providing researchers with data to assist their selection of a suitable isolation method.


Assuntos
Fracionamento Celular/métodos , Células Endoteliais/química , Vesículas Extracelulares/química , Biomarcadores/análise , Centrifugação/métodos , Precipitação Química , DNA/análise , Células Endoteliais/virologia , Vesículas Extracelulares/virologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/isolamento & purificação , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas/análise , RNA/análise
20.
Water Sci Technol ; 81(8): 1715-1722, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32644963

RESUMO

Most models of sedimentation contain the nonlinear hindered-settling flux function. If one assumes ideal conditions and no compression, then there exist several theoretically possible ways of identifying a large portion of the flux function from only one experiment by means of formulas derived from the theory of solutions of partial differential equations. Previously used identification methods and recently published such, which are based on utilizing conical vessels or centrifuges, are reviewed and compared with synthetic data (simulated experiments). This means that the identification methods are evaluated from a theoretical viewpoint without experimental errors or difficulties. The main contribution of the recent methods reviewed is that they, in theory, can identify a large portion of the flux function from a single experiment, in contrast to the traditional method that provides one point on the flux curve from each test. The new methods lay the foundation of rapid flux identification; however, experimental procedures need to be elaborated.


Assuntos
Modelos Teóricos , Centrifugação
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