Year: 2022
Type: article
Abstract: Recent advances in flow cytometry have allowed high-dimensional characterization of biological phenomena, enabling breakthroughs in a multitude of fields. Despite the appreciation of the unique properties of antigens and fluorophores in high-parameter panel design, staining conditions are often standardized for short surface stains, regardless of antibody affinity or antigen accessibility. Here, we demonstrate how increasing antibody incubation times can lead to substantial improvements in sensitivity, maintaining specificity, and reducing background, while also significantly reducing the costs of high-parameter cytometry panels. Furthermore, overnight staining reduces the influence of interexperimental variability, assisting accurate pooling over experiments over extended time courses. We provide guidance on how to optimize staining conditions for diverse antigens, including how different fixation strategies can affect epitope accessibility. Overnight staining can thus substantially improve the resolution, repeatability, and cost-effectiveness of high-parameter cytometry. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.
Source: Current Protocols
DOI: 10.1002/cpz1.589
Language: English
Subfield: Radiology, Nuclear Medicine and Imaging
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $4,750
Awards: G1990583613, Cytek Aurora Spectral Analyser: essential investment to expand the Babraham Institute Flow Cytometry capabilities, enabling pioneering bioscience, G4537595325, Develop a mechanistic understanding of how transcriptional and posttranscriptional regulation of gene expression is coordinated by signal transduction pathways., G6160911136, Resolving eosinophil and inflammatory heterogeneity in chronic allergic airway disease for safer, effective and lasting precision therapies, Immune homeostasis and the immune response, G7802970066
OpenAlex ID: W4308979128