Screening protocol for the identification of modulators by immunofluorescent cell‐based assay
| Autor: | Joaquín Pastor, Marinela Méndez-Pertuz, Sonia Sánchez Martínez, Carmen Blanco-Aparicio, Manuel Urbano-Cuadrado, Elena Gómez-Casero, Diego Megías, Maria A. Blasco, Javier Klett |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Cellular activity
Computer science High-throughput screening Drug Evaluation Preclinical Fluorescent Antibody Technique Computational biology 01 natural sciences Biochemistry Antibodies law.invention Small Molecule Libraries Confocal microscopy law Cell Line Tumor Drug Discovery Humans Telomeric Repeat Binding Protein 1 Fluorescent Dyes Pharmacology Protocol (science) Microscopy Confocal 010405 organic chemistry Optical Imaging Organic Chemistry Reproducibility of Results Telomere High-Throughput Screening Assays 0104 chemical sciences 010404 medicinal & biomolecular chemistry Identification (information) High-content screening Molecular Medicine Target protein Cell based |
| Zdroj: | Chemical Biology & Drug Design. 95:66-78 |
| ISSN: | 1747-0285 1747-0277 |
| DOI: | 10.1111/cbdd.13616 |
| Popis: | High-throughput assays are a common strategy for the identification of compounds able to modulate a certain cellular activity. Here, we show an automatized analysis platform for the quantification of nuclear foci as inhibitory effect of compounds on a target protein labeled by fluorescent antibodies. Our experience led us to a fast analysis platform that combines cell-based assays, high-content screening, and confocal microscopy, with an automatic and user-friendly statistical analysis of plate-based assays including positive and negative controls, able to identify inhibitory effect of compounds tested together with the Z-prime and Window of individual plate-based assays to assess the reliability of the results. The platform integrates a web-based tool implemented in Pipeline Pilot and R, and allows computing the inhibition values of different parameters obtained from the high-content screening and confocal microscopy analysis. This facilitates the exploration of the results using the different parameters, providing information at different levels as the number of foci observed, the sum of intensity of foci, area of foci, etc, the detection and filtering of outliers over the assay plate, and finally providing a set of statistics of the parameters studied together with a set of plots that we believe significantly helps to the interpretation of the assay results. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text