PROCESS FOR MEASUREMENT OF LIPID PEROXIDATION IN ADHERENT MAMMALIAN CELL LINES

Abstract

This invention provides a process for measuring lipid peroxidation in mammalian cell cultures using an assay kit. The kit includes reagents for preparing cell lysates and quantifying malondialdehyde levels. Designed for reliability and ease of use, the protocol allows for efficient oxidative stress assessment in laboratory settings.

Specification

Description:FIELD OF THE INVENTION
This invention relates to laboratory diagnostics, specifically a process for quantifying lipid peroxidation in in vitro adherent mammalian cell lines. The invention offers a streamlined, efficient, and reproducible method for assessing oxidative stress by measuring malondialdehyde (MDA), a by-product of lipid peroxidation, within cell cultures.
BACKGROUND OF THE INVENTION
Lipid peroxidation, a marker of oxidative stress, plays a significant role in various disease processes, including cancer, diabetes, and cardiovascular diseases. Typically, existing protocols for measuring lipid peroxidation focus on tissue samples rather than in vitro cell lines, resulting in limited options for adherent cell assessment. Current methods also lack standardization, leading to variability in results. This invention provides a modified protocol specifically tailored to in vitro adherent mammalian cell lines, allowing for accurate, efficient, and reproducible quantification of lipid peroxidation using an assay designed to measure malondialdehyde (MDA) levels. The assay kit is user-friendly, requires minimal specialized training, and delivers results within a few hours, making it ideal for both research and clinical laboratories.
SUMMARY OF THE INVENTION
This summary is provided to introduce a selection of concepts, in a simplified format, that are further described in the detailed description of the invention.
This summary is neither intended to identify key or essential inventive concepts of the invention and nor is it intended for determining the scope of the invention.
To further clarify advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings.
The invention offers a lipid peroxidation assay kit for quantifying malondialdehyde (MDA) levels in adherent mammalian cell lines. The kit includes reagents, instructions for sample preparation, and a standardized protocol that ensures consistent and reproducible results. Key components are MDA bis(Dimethyl Acetal), Reagent A (Sodium acetate), Reagent B (Thiobarbituric acid), and Reagent C (Sodium dodecyl sulfate), each optimized to interact within cell lysates prepared from cultured cells. The assay process includes preparation of the cell lysate, reaction with assay reagents, and analysis of absorbance to determine MDA concentrations. This kit addresses the limitations of existing methods by providing a reliable, efficient, and easy-to-follow protocol for lipid peroxidation measurement in mammalian cell lines.
DETAILED DESCRIPTION OF THE INVENTION
The detailed description of various exemplary embodiments of the disclosure is described herein with reference to the accompanying drawings. It should be noted that the embodiments are described herein in such details as to clearly communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the present disclosure as defined by the appended claims.
It is also to be understood that various arrangements may be devised that, although not explicitly described or shown herein, embody the principles of the present disclosure. Moreover, all statements herein reciting principles, aspects, and embodiments of the present disclosure, as well as specific examples, are intended to encompass equivalents thereof.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments. As used herein, the singular forms "a"," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises," "comprising," "includes" and/or "including," when used herein, specify the presence of stated features, integers, steps, operations, elements and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and/or groups thereof.
It should also be noted that in some alternative implementations, the functions/acts noted may occur out of the order noted in the figures. For example, two figures shown in succession may, in fact, be executed concurrently or may sometimes be executed in the reverse order, depending upon the functionality/acts involved.
In addition, the descriptions of "first", "second", "third", and the like in the present invention are used for the purpose of description only, and are not to be construed as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Thus, features defining "first" and "second" may include at least one of the features, either explicitly or implicitly.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
The lipid peroxidation measurement process for adherent mammalian cell lines begins with the preparation of MDA standards and reagents. The primary reagent, MDA bis(Dimethyl Acetal), is prepared as a stock solution, which is then diluted into sub-stocks for use in the assay. Cell lysates are prepared by washing confluent cells with PBS, trypsinizing them, and collecting the resulting cell pellet. The pellet is resuspended in a lysis buffer containing RIPA and protease inhibitors to preserve the integrity of cellular components.
Following lysis, the supernatant is prepared by centrifuging the lysate, and a reaction mixture is created by combining the lysate with assay reagents. The reagents include sodium acetate, thiobarbituric acid, and sodium dodecyl sulfate, which react with MDA to produce a chromogenic complex measurable via absorbance. The reaction mixture is incubated at 90°C for an hour to facilitate the development of the chromogenic signal. After cooling, the solution is centrifuged, and the supernatant is transferred to a 96-well plate for absorbance reading at 532 nm. A standard curve is generated by plotting absorbance readings of known MDA concentrations, allowing for the accurate quantification of lipid peroxidation in the sample.
The assay protocol is designed for ease of use, providing reliable results within 2-3 hours and accommodating multiple samples in 6-well culture plates simultaneously. This kit addresses the need for a standardized, reproducible method for measuring lipid peroxidation in in vitro cell lines, enhancing the reliability of oxidative stress assessments in cellular research. , Claims:1. A process for measuring lipid peroxidation in adherent mammalian cell lines, utilizing a reagent kit comprising MDA bis(Dimethyl Acetal), sodium acetate, thiobarbituric acid, and sodium dodecyl sulfate.
2. The process as claimed in Claim 1, wherein cell lysates are prepared from mammalian cell cultures through washing, trypsinization, and lysis in RIPA buffer with protease inhibitors.
3. The process as claimed in Claim 1, wherein the assay reaction involves combining cell lysate with assay reagents and incubating at 90°C for color development.
4. The process as claimed in Claim 1, wherein the resulting reaction mixture is centrifuged and the supernatant analyzed via absorbance at 532 nm to quantify malondialdehyde levels.
5. The process as claimed in Claim 1, wherein a standard curve is generated using known MDA concentrations for accurate measurement of lipid peroxidation.
6. The process as claimed in Claim 1, designed to be performed within 2-3 hours, allowing for efficient and reproducible quantification of oxidative stress in cell lines.
7. The process as claimed in Claim 1, configured to analyze multiple samples simultaneously using a 96-well plate format, enhancing laboratory throughput.
8. The process as claimed in Claim 1, providing a standardized and reproducible protocol for lipid peroxidation measurement, addressing the limitations of existing tissue-based assays.

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