Dataset Information

A high-throughput method for the quantification of iron saturation in lactoferrin preparations.

ABSTRACT: Lactoferrin is considered as a part of the innate immune system that plays a crucial role in preventing bacterial growth, mostly via an iron sequestration mechanism. Recent data show that bovine lactoferrin prevents late-onset sepsis in preterm very low birth weight neonates by serving as an iron chelator for some bacterial strains; thus, it is very important to control the iron saturation level during diet supplementation. An accurate estimation of lactoferrin iron saturation is essential not only because of its clinical applications but also for a wide range of biochemical experiments. A comprehensive method for the quantification of iron saturation in lactoferrin preparations was developed to obtain a calibration curve enabling the determination of iron saturation levels relying exclusively on the defined ratio of absorbances at 280 and 466 nm (A(280/466)). To achieve this goal, selected techniques such as spectrophotometry, ELISA, and ICP-MS were combined. The ability to obtain samples of lactoferrin with determination of its iron content in a simple and fast way has been proven to be very useful. Furthermore, a similar approach could easily be implemented to facilitate the determination of iron saturation level for other metalloproteins in which metal binding results in the appearance of a distinct band in the visible part of the spectrum.

SUBMITTER: Majka G

PROVIDER: S-EPMC3656221 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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A high-throughput method for the quantification of iron saturation in lactoferrin preparations.

Majka Grzegorz G Śpiewak Klaudyna K Kurpiewska Katarzyna K Heczko Piotr P Stochel Grażyna G Strus Magdalena M Brindell Małgorzata M

Analytical and bioanalytical chemistry 20130421 15

Lactoferrin is considered as a part of the innate immune system that plays a crucial role in preventing bacterial growth, mostly via an iron sequestration mechanism. Recent data show that bovine lactoferrin prevents late-onset sepsis in preterm very low birth weight neonates by serving as an iron chelator for some bacterial strains; thus, it is very important to control the iron saturation level during diet supplementation. An accurate estimation of lactoferrin iron saturation is essential not o ...[more]

PMID: 23604471

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Project description:Multicellular spheroids embedded in 3D hydrogels are prominent in vitro models for 3D cell invasion. Yet, quantification methods for spheroid cell invasion that are high throughput, objective and accessible are still lacking. Variations in spheroid sizes and the shapes of the cells within render it difficult to objectively assess invasion extent. The goal of this work is to develop a high-throughput quantification method of cell invasion into 3D matrices that minimizes sensitivity to initial spheroid size and cell spreading and provides precise integrative directionally-dependent metrics of invasion. By analyzing images of fluorescent cell nuclei, invasion metrics are automatically calculated at the pixel level. The initial spheroid boundary is segmented and automated calculations of the nuclear pixel distances from the initial boundary are used to compute common invasion metrics (i.e., the change in invasion area, mean distance) for the same spheroid at a later timepoint. We also introduce the area moment of inertia as an integrative metric of cell invasion that considers the invasion area as well as the pixel distances from the initial spheroid boundary. Further, we show that principal component analysis can be used to quantify the directional influence of a stimuli to invasion (e.g., due to a chemotactic gradient or contact guidance). To demonstrate the power of the analysis for cell types with different invasive potentials and the utility of this method for a variety of biological applications, the method is used to analyze the invasiveness of five different cell types. In all, implementation of this high throughput quantification method results in consistent and objective analysis of 3D multicellular spheroid invasion. We provide the analysis code in both MATLAB and Python languages as well as a GUI for ease of use for researchers with a range of computer programming skills and for applications in a variety of biological research areas such as wound healing and cancer metastasis.

Project description:Multicellular spheroids embedded in 3D hydrogels are prominent in vitro models for 3D cell invasion. Yet, quantification methods for spheroid cell invasion that are high-throughput, objective and accessible are still lacking. Variations in spheroid sizes and the shapes of the cells within render it difficult to objectively assess invasion extent. The goal of this work is to develop a high-throughput quantification method of cell invasion into 3D matrices that minimizes sensitivity to initial spheroid size and cell spreading and provides precise integrative directionally-dependent metrics of invasion. By analyzing images of fluorescent cell nuclei, invasion metrics are automatically calculated at the pixel level. The initial spheroid boundary is segmented and automated calculations of the nuclear pixel distances from the initial boundary are used to compute common invasion metrics (i.e., the change in invasion area, mean distance) for the same spheroid at a later timepoint. We also introduce the area moment of inertia as an integrative metric of cell invasion that considers the invasion area as well as the pixel distances from the initial spheroid boundary. Further, we show that principal component analysis can be used to quantify the directional influence of a stimuli to invasion (e.g., due to a chemotactic gradient or contact guidance). To demonstrate the power of the analysis for cell types with different invasive potentials and the utility of this method for a variety of biological applications, the method is used to analyze the invasiveness of five different cell types. In all, implementation of this high-throughput quantification method results in consistent and objective analysis of 3D multicellular spheroid invasion. We provide the analysis code in both MATLAB and Python languages as well as a GUI for ease of use for researchers with a range of computer programming skills and for applications in a variety of biological research areas such as wound healing and cancer metastasis.

Dataset Information

A high-throughput method for the quantification of iron saturation in lactoferrin preparations.

Publications

A high-throughput method for the quantification of iron saturation in lactoferrin preparations.

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