Dataset Information

Quantitative determination of fluorescence labeling implemented in cell cultures.

ABSTRACT:

Background

Labeling efficiency is a crucial parameter in fluorescence applications, especially when studying biomolecular interactions. Current approaches for estimating the yield of fluorescent labeling have critical drawbacks that usually lead them to be inaccurate or not quantitative.

Results

We present a method to quantify fluorescent-labeling efficiency that addresses the critical issues marring existing approaches. The method operates in the same conditions of the target experiments by exploiting a ratiometric evaluation with two fluorophores used in sequential reactions. We show the ability of the protocol to extract reliable quantification for different fluorescent probes, reagents concentrations, and reaction timing and to optimize labeling performance. As paradigm, we consider the labeling of the membrane-receptor TrkA through 4'-phosphopantetheinyl transferase Sfp in living cells, visualizing the results by TIRF microscopy. This investigation allows us to find conditions for demanding single and multi-color single-molecule studies requiring high degrees of labeling.

Conclusions

The developed method allows the quantitative determination and the optimization of staining efficiency in any labeling strategy based on stable reactions.

SUBMITTER: Schirripa Spagnolo C

PROVIDER: S-EPMC10496409 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Publications

Quantitative determination of fluorescence labeling implemented in cell cultures.

Schirripa Spagnolo Chiara C Moscardini Aldo A Amodeo Rosy R Beltram Fabio F Luin Stefano S

BMC biology 20230912 1

<h4>Background</h4>Labeling efficiency is a crucial parameter in fluorescence applications, especially when studying biomolecular interactions. Current approaches for estimating the yield of fluorescent labeling have critical drawbacks that usually lead them to be inaccurate or not quantitative.<h4>Results</h4>We present a method to quantify fluorescent-labeling efficiency that addresses the critical issues marring existing approaches. The method operates in the same conditions of the target exp ...[more]

PMID: 37697318

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Project description:Phagocytosis is series of steps where the pathogens and the immune cells interact during an invasion. This starts with the adhesion process between the host and pathogen cells, and is followed by the engulfment of the pathogens. Many analytical methods that are applied to characterize phagocytosis based on imaging the host-pathogen confrontation assays rely on the fluorescence labeling of cells. However, the potential effect of the membrane labeling on the quantitative results of the confrontation assays has not been studied in detail. In this study, we determine whether the fluorescence labeling processes themselves influence the results of the phagocytosis measurements. Here, alveolar macrophages, which form one of the most important compartments of the innate immune system, were used as an example of host cells, whereas Aspergillus fumigatus and Lichtheimia corymbifera that cause aspergillosis and mucormycosis, respectively, were studied as examples for pathogens. At first, our study investigated the importance of the sequence of steps of the fixation process when preparing the confrontation assay sample for microscopy studies. Here we showed that applying the fixation agent before the counter-staining causes miscalculations during the determination of the phagocytic measures. Furthermore, we also found that staining the macrophages with various concentrations of DID, as a typical membrane label, in most cases altered the capability of macrophages to phagocytose FITC-stained A. fumigatus and L. corymbifera spores in comparison with unlabeled macrophages. This effect of the DID staining showed a differential character dependent upon the labeling status and the specific type of pathogen. Moreover, labeling the spores of A. fumigatus and L. corymbifera with FITC increased the phagocytic measures during confrontation with unlabeled macrophages when compared to label-free spores. Overall, our study confirms that the staining process itself may significantly manipulate the quantitative outcome of the confrontation assay. As a result of our study, we also developed a user-friendly image analysis tool that analyses confrontation assays both with and without fluorescence labeling of the host cells and of the pathogens. Our image analysis algorithm saves experimental work effort and time, provides more precise results when calculating the phagocytic measures, and delivers a convenient analysis tool for the biologists to monitor host-pathogen interactions as they happen without the artifacts that fluorescence labeling imposes on biological interactions.

Project description:BackgroundWith increasing pollution, herbicide application and interest in plant phenotyping, sensors capturing early responses to toxic stress are demanded for screening susceptible or resistant plant varieties. Standard toxicity tests on plants are laborious, demanding in terms of space and material, and the measurement of growth-inhibition based endpoints takes relatively long time. The aim of this work was to explore the potential of photoautotrophic cell suspension cultures for high-throughput early toxicity screening based on imaging techniques. The investigation of the universal potential of fluorescence imaging methods involved testing of three toxicants with different modes of action (DCMU, glyphosate and chromium).ResultsThe increased pace of testing was achieved by using non-destructive imaging methods-multicolor fluorescence (MCF) and chlorophyll fluorescence (ChlF). These methods detected the negative effects of the toxicants earlier than it was reflected in plant growth inhibition (decrease in leaf area and final dry weight). Moreover, more subtle and transient effects not resulting in growth inhibition could be detected by fluorescence. The pace and sensitivity of stress detection was further enhanced by using photoautotrophic cell suspension cultures. These reacted sooner, more pronouncedly and to lower concentrations of the tested toxicants than the plants. Toxicant-specific stress signatures were observed as a combination of MCF and ChlF parameters and timing of the response. Principal component analysis was found to be useful for reduction of the collected multidimensional data sets to a few informative parameters allowing comparison of the toxicant signatures.ConclusionsPhotoautotrophic cell suspension cultures have proved to be useful for rapid high-throughput screening of toxic stress and display a potential for employment as an alternative to tests on whole plants. The MCF and ChlF methods are capable of distinguishing early stress signatures of at least three different modes of action.

Dataset Information

Quantitative determination of fluorescence labeling implemented in cell cultures.

Background

Results

Conclusions

Publications

Quantitative determination of fluorescence labeling implemented in cell cultures.

Similar Datasets

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