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Pitfalls and solutions in assaying anandamide transport in cells.

ABSTRACT: Nonspecific binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of conflicting data on the uptake and release of this lipophilic substance. Herein, we propose an improved method to assay anandamide transport, by using glass slides (i.e., coverslips) as physical support to grow cells. Although the results obtained using plastic do not differ significantly from those obtained using glass, the new procedure has the advantage of being faster, simpler, and more accurate. In fact, the lack of aspecific adsorption of anandamide to the glass surface yields a lower background and a higher precision and accuracy in determining transport kinetics, especially for the export process. Remarkably, the kinetic parameters of anandamide uptake obtained with the old and the new procedures may be similar or different depending on the cell type, thus demonstrating the complexity of the interference of plastic on the transport process. In addition, the novel procedure is particularly suitable for visualization and measurement of anandamide transport in intact cells by using a biotinylated derivative in confocal fluorescence microscopy.

SUBMITTER: Oddi S

PROVIDER: S-EPMC2903826 | biostudies-other | 2010 Aug

REPOSITORIES: biostudies-other

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Pitfalls and solutions in assaying anandamide transport in cells.

Oddi Sergio S Fezza Filomena F Catanzaro Giuseppina G De Simone Chiara C Pucci Mariangela M Piomelli Daniele D Finazzi-Agrò Alessandro A Maccarrone Mauro M

Journal of lipid research 20100506 8

Nonspecific binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of conflicting data on the uptake and release of this lipophilic substance. Herein, we propose an improved method to assay anandamide transport, by using glass slides (i.e., coverslips) as physical support to grow cells. Although the results obtained using plastic do not differ significantly from those obtained using glass, the new procedure ...[more]

PMID: 20447929

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Project description:BackgroundSnakebites are a public health problem in Nicaragua: it is a tropical developing country, venomous snakes are present and there are reports of snakebites treated both in the formal and informal health care system. We aimed to produce an incidence map using data reported by the health care system that would be used to allocate resources. However, this map may suffer from case detection bias and decisions based on this map will neglect snakebite victims who do not receive healthcare. To avoid this error, we try to identify where underreporting is likely based on available information.Method and findingsThe Nicaraguan municipalities are categorized by precipitation, altitude and geographical location into regions of assumed homogenous snake prevalence. Socio-economic and healthcare variables hypothesized to be related to underreporting of snakebites are aggregated into an index. The environmental region variable, the underreporting index and three demographic variables (rurality, sex and age distribution) are entered in a Poisson regression model of municipality-level snakebite incidence. In this model, the underreporting index is non-linearly associated with snakebite incidence, a finding we attribute to underreporting in the most deprived municipalities. The municipalities with the worst scoring on the underreporting index and those with combined low reported incidence and large rural population are identified as likely underreporting. 3,286 snakebite cases were reported in 2005-2009, corresponding to a 5-year incidence of 56 bites per 100,000 inhabitants (municipality range: 0-600 cases per 100,000 inhabitants).ConclusionsUsing publicly available data, we identified areas likely to be underreporting snakebites and highlighted these areas instead of leaving them "white" on the incidence map. The effects of the case detection bias on the distribution of resources against snakebites could decrease. Although not yet verified empirically, our study provides an example of how snake bite epidemiology may be investigated in similar settings worldwide at a low cost.

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Project description:The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for quantifying DNA available for PCR.

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Pitfalls and solutions in assaying anandamide transport in cells.

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Pitfalls and solutions in assaying anandamide transport in cells.

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