Effects of harvesting and extraction methods on metabolite recovery from adherently growing mammalian cells

2020 ◽  
Vol 12 (19) ◽  
pp. 2491-2498
Author(s):  
Yun Luo ◽  
Ningbo Geng ◽  
Baoqin Zhang ◽  
Jiping Chen ◽  
Haijun Zhang
Keyword(s):  
Sample Preparation ◽  
Mammalian Cells ◽  
Extraction Methods ◽  
Sample Processing ◽  
Cell Harvesting ◽  
A Cell ◽  
Cell Sample ◽  
Metabolite Extraction

We compare the efficiencies of different cell harvesting methods and metabolite extraction methods in sample preparation procedures and provide a cell sample processing protocol which focuses on maximizing metabolite recovery ranging from polar to lipidic ones.

scholarly journals Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 566 ◽  
Author(s):  
Siemon Ng ◽  
Cassandra Braxton ◽  
Marc Eloit ◽  
Szi Feng ◽  
Romain Fragnoud ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Nucleic Acids ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Virus Detection ◽  
Extraction Methods ◽  
Control Measures ◽  
Acid Extraction ◽  
Library Preparation ◽  
Sample Processing

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.

scholarly journals Grass Material as a Modern Process Standard for 14C Analysis of n-Alkanes

Radiocarbon ◽  
2016 ◽  
Vol 58 (3) ◽  
pp. 445-458 ◽  
Author(s):  
L M Cisneros-Dozal ◽  
X Xu ◽  
C Bryant ◽  
E J Pearson ◽  
J A J Dungait
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Sample Preparation ◽  
Extraction Methods ◽  
Chemical Extraction ◽  
Sample Processing ◽  
Elution Time ◽  
Modern Process ◽  
Chemical Extraction Methods ◽  
Suitable Process

AbstractOne of the difficulties in reporting accurate radiocarbon results from compound-specific radiocarbon analysis (CSRA) is the lack of suitable process standard materials to correct for the amount and 14C content of carbon added during extensive sample processing. We evaluated the use of n-alkanes extracted from modern grass material (1.224±0.006 fraction modern) as process standards for CSRA. The n-alkanes were isolated using preparative capillary gas chromatography (PCGC) from two independent chemical extraction methods applied to the grass. Since this was our first assessment of the 14C content of the grass n-alkanes, we corrected for extraneous carbon derived from PCGC isolation using commercially available single compounds of modern and 14C-free content. Results were consistent across the two extraction methods showing that the C29n-alkane has a fraction modern value that is within 1σ of the bulk value of the grass while C31n-alkane and less abundant n-alkanes have values within 2σ of the bulk value of the grass. C29 and C31n-alkanes were the most abundant n-alkanes in the grass and, as such, the more feasible for collection of sufficient amounts of carbon for accelerator mass spectrometry (AMS) analysis. Our results suggest that choosing a grass n-alkane with an elution time closest to that of the unknowns may be advisable due to possibly greater effect from GC column bleed (14C-free) at later elution times. We conclude that C29 and C31n-alkanes in modern grass of known 14C content can be used as in-house standards to correct for the addition of 14C-free carbon during sample preparation for 14C analysis of n-alkanes.

scholarly journals Experimental Design and Sample Preparation in Forest Tree Metabolomics

Metabolites ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 285 ◽  
Author(s):  
Ana M. Rodrigues ◽  
Ana I. Ribeiro-Barros ◽  
Carla António
Keyword(s):  
Experimental Design ◽  
Sample Preparation ◽  
Forest Tree ◽  
Sample Processing ◽  
Technical Aspects ◽  
Plant Metabolomics ◽  
Biological Interpretation ◽  
Laboratory Procedures ◽  
Key Steps ◽  
Metabolite Extraction

Appropriate experimental design and sample preparation are key steps in metabolomics experiments, highly influencing the biological interpretation of the results. The sample preparation workflow for plant metabolomics studies includes several steps before metabolite extraction and analysis. These include the optimization of laboratory procedures, which should be optimized for different plants and tissues. This is particularly the case for trees, whose tissues are complex matrices to work with due to the presence of several interferents, such as oleoresins, cellulose. A good experimental design, tree tissue harvest conditions, and sample preparation are crucial to ensure consistency and reproducibility of the metadata among datasets. In this review, we discuss the main challenges when setting up a forest tree metabolomics experiment for mass spectrometry (MS)-based analysis covering all technical aspects from the biological question formulation and experimental design to sample processing and metabolite extraction and data acquisition. We also highlight the importance of forest tree metadata standardization in metabolomics studies.

Development of a C12Im-Cl-assisted method for rapid sample preparation in proteomic application

2021 ◽  
Author(s):  
Chang Liu ◽  
Xiaoxia Si ◽  
Shumei Yan ◽  
Xinyuan Zhao ◽  
Xiaohong Qian ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Extraction ◽  
Sample Processing ◽  
Processing Methods ◽  
Proteomic Analyses

Chromatography and mass spectrometry (MS) techniques have greatly improved the power of proteomic analyses. However, sample processing methods, including protein extraction and digestion, before MS remain as bottlenecks in the...

Sterol and lipid trafficking in mammalian cells

2006 ◽  
Vol 34 (3) ◽  
pp. 335-339 ◽  
Author(s):  
F.R. Maxfield ◽  
M. Mondal
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Vesicular Transport ◽  
Cholesterol Content ◽  
Cellular Functions ◽  
Lipid Trafficking ◽  
Sterol Transport ◽  
A Cell ◽  
Asymmetrically Distributed

The pathways involved in the intracellular transport and distribution of lipids in general, and sterols in particular, are poorly understood. Cholesterol plays a major role in modulating membrane bilayer structure and important cellular functions, including signal transduction and membrane trafficking. Both the overall cholesterol content of a cell, as well as its distribution in specific organellar membranes are stringently regulated. Several diseases, many of which are incurable at present, have been characterized as results of impaired cholesterol transport and/or storage in the cells. Despite their importance, many fundamental aspects of intracellular sterol transport and distribution are not well understood. For instance, the relative roles of vesicular and non-vesicular transport of cholesterol have not yet been fully determined, nor are the non-vesicular transport mechanisms well characterized. Similarly, whether cholesterol is asymmetrically distributed between the two leaflets of biological membranes, and if so, how this asymmetry is maintained, is poorly understood. In this review, we present a summary of the current understanding of these aspects of intracellular trafficking and distribution of lipids, and more specifically, of sterols.

scholarly journals The Protective Role of Dormant Origins in Response to Replicative Stress

2018 ◽  
Vol 19 (11) ◽  
pp. 3569 ◽  
Author(s):  
Lilas Courtot ◽  
Jean-Sébastien Hoffmann ◽  
Valérie Bergoglio
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Replication Timing ◽  
Protective Role ◽  
Entire Genome ◽  
Replicative Stress ◽  
A Cell ◽  
The Many

Genome stability requires tight regulation of DNA replication to ensure that the entire genome of the cell is duplicated once and only once per cell cycle. In mammalian cells, origin activation is controlled in space and time by a cell-specific and robust program called replication timing. About 100,000 potential replication origins form on the chromatin in the gap 1 (G1) phase but only 20–30% of them are active during the DNA replication of a given cell in the synthesis (S) phase. When the progress of replication forks is slowed by exogenous or endogenous impediments, the cell must activate some of the inactive or “dormant” origins to complete replication on time. Thus, the many origins that may be activated are probably key to protect the genome against replication stress. This review aims to discuss the role of these dormant origins as safeguards of the human genome during replicative stress.

scholarly journals Benchtop-compatible sample processing workflow for proteome profiling of < 100 mammalian cells

2018 ◽  
Vol 411 (19) ◽  
pp. 4587-4596 ◽  
Author(s):  
Kerui Xu ◽  
Yiran Liang ◽  
Paul D. Piehowski ◽  
Maowei Dou ◽  
Kaitlynn C. Schwarz ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Sample Processing ◽  
Proteome Profiling

scholarly journals Serotonin and dopamine modulate aging in response to food perception and availability

2021 ◽  
Author(s):  
Hillary A. Miller ◽  
Shijiao Huang ◽  
Megan L. Schaller ◽  
Elizabeth S. Dean ◽  
Angela M. Tuckowski ◽  
...  
Keyword(s):  
Dopamine Receptors ◽  
Signaling Pathway ◽  
Dopamine Receptor ◽  
Dietary Restriction ◽  
Mammalian Cells ◽  
Serotonin Receptor ◽  
Enteric Neuron ◽  
C Elegans ◽  
Food Perception ◽  
A Cell

AbstractAn organism’s ability to perceive and respond to changes in its environment is crucial for its health and survival. Here we reveal how the most well-studied longevity intervention, dietary restriction (DR), acts in-part through a cell non-autonomous signaling pathway that is inhibited by the perception of attractive smells. Using an intestinal reporter for a key gene induced by DR but suppressed by attractive smells, we identify three compounds that block food perception in C. elegans, thereby increasing longevity as DR mimetics. These compounds clearly implicate serotonin and dopamine in limiting lifespan in response to food perception. We further identify an enteric neuron in this pathway that signals through the serotonin receptor 5-HT1A/ser-4 and dopamine receptor DRD2/dop-3. Aspects of this pathway are conserved in D. melanogaster and mammalian cells. Thus, blocking food perception through antagonism of serotonin or dopamine receptors is a plausible approach to mimic the benefits of dietary restriction.

Smartphone integrated optoelectrowetting (SiOEW) for on-chip sample processing and microscopic detection of water quality

Lab on a Chip ◽  
2018 ◽  
Vol 18 (3) ◽  
pp. 532-539 ◽  
Author(s):  
Dongyue Jiang ◽  
Seunguk Lee ◽  
Sung Woo Bae ◽  
Sung-Yong Park
Keyword(s):  
Water Quality ◽  
Sample Preparation ◽  
Low Cost ◽  
Sample Processing ◽  
Microscopic Detection ◽  
Chip Sample ◽  
On Chip

We present a smartphone integrated optoelectrowetting (SiOEW) device as a low-cost, portable tool for on-chip sample preparation and microscopic detection of water quality.

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