UEMU: Upgrade Proposal for the Metabolism and Flow Cytometry Facility of the Institute of Biology of Unicamp.

• Grant number: 23/17089-6
• Support Opportunities: Research Infrastructure Program - Small Medium Sized
• Start date: September 01, 2024
• End date: August 31, 2027
• Field of knowledge: Biological Sciences - Immunology - Cellular Immunology
• Principal Investigator: Pedro Manoel Mendes de Moraes Vieira
• Grantee: Pedro Manoel Mendes de Moraes Vieira
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated researchers: Carlos Henrique Grossi Sponton ; Fabio Trindade Maranhão Costa

Abstract

The present project aims to acquire the Agilent Seahorse XF Imaging and QX200 Droplet Digital PCR System equipment to enhance and strengthen the capabilities of the multi-user Facility for metabolism and flow cytometry at the Institute of Biology of Unicamp. Currently, the Facility, in the metabolism area, has two equipment pieces acquired with FAPESP funding for real-time metabolic studies (Seahorse Analyzer XFe24 and Seahorse Analyzer XFe96). The Agilent Seahorse XF Imaging is a multifunctional instrument that integrates bright-field microscopy, fluorescence, and microplate reading for the integration and normalization of data obtained from the Seahorse Analyzer. Data normalization is essential for the validity of any experiment, especially when there are variations in the sample quantity. In real-time cellular metabolic analysis using Agilent Seahorse XF technology, the cell quantity per well is the most accepted form as a reference value for data normalization. This can be determined either by direct cell counting or indirectly by measuring biomolecules such as total proteins or genomic DNA. The Agilent Seahorse XF Normalization Imaging system integrates XF technology with a Agilent Seahorse XF Imaging Multi-Mode reader. In this configuration, the Agilent Seahorse XF Imaging is controlled by the new Agilent Seahorse XF Imaging and Cell Counting software, offering a simplified, automated, and validated solution for cell counting based on microscopic images. This enables: i) the in situ application of a membrane-permeable nuclear dye (e.g., Hoechst 33342, mitogreen), ii) automated imaging and optimized cell counting for XF assays, and iii) synchronization of images and cell counting in XF results files in the Wave software. This provides an efficient and reliable normalization method, offering all XF Analyzer users a continuous, fast, reliable, and updated workflow in line with current requirements in metabolic studies. Digital PCR is an innovative technology that provides ultrasensitive detection of nucleic acids and absolute quantification. It is highly effective for low-abundance targets, such as allelic or structural variants, which are below the detection level of other platforms. Digital PCR strategies have great potential and will be successfully applied for: (i) measuring biomarkers in various clinical scenarios; (ii) genotyping assays and metabolic modulations of drugs for detecting polymorphisms in genes encoding various drug-metabolizing enzymes (DMEs) and associated transport proteins; (iii) accurate quantification and qualification of NGS libraries without the use of a standard curve, among other applications. (AU)