Back to Search
Start Over
FixNCut: A Practical Guide to Sample Preservation by Reversible Fixation for Single Cell Assays.
- Source :
-
Bio-protocol [Bio Protoc] 2024 Sep 05; Vol. 14 (17), pp. e5063. Date of Electronic Publication: 2024 Sep 05 (Print Publication: 2024). - Publication Year :
- 2024
-
Abstract
- The quality of standard single-cell experiments often depends on the immediate processing of cells or tissues post-harvest to preserve fragile and vulnerable cell populations, unless the samples are adequately fixed and stored. Despite the recent rise in popularity of probe-based and aldehyde-fixed RNA assays, these methods face limitations in species and target availability and are not suitable for immunoprofiling or assessing chromatin accessibility. Recently, a reversible fixation strategy known as FixNCut has been successfully deployed to separate sampling from downstream applications in a reproducible and robust manner, avoiding stress or necrosis-related artifacts. In this article, we present an optimized and robust practical guide to the FixNCut protocol to aid the end-to-end adaptation of this versatile method. This protocol not only decouples tissue or cell harvesting from single-cell assays but also enables a flexible and decentralized workflow that unlocks the potential for single-cell analysis as well as unconventional study designs that were previously considered unfeasible. Key features • Reversible fixation: Preserves cellular and molecular structures with the option to later reverse the fixation for downstream applications, maintaining cell integrity • Compatibility with single-cell assays: Supports single-cell genomic assays such as scRNA-seq and ATAC-seq, essential for high-resolution analysis of cell function and gene expression • Flexibility in sample handling: Allows immediate fixation post-collection, decoupling sample processing from analysis, beneficial in settings where immediate processing is impractical • Preservation of RNA and DNA integrity: Effectively preserves RNA and DNA, reducing degradation to ensure accurate transcriptomic and genomic profiling • Suitability for various biological samples: Applicable to a wide range of biological samples, including tissues and cell suspensions, whether freshly isolated or post-dissociated • Enables multi-center studies: Facilitates collaborative research across multiple centers by allowing sample fixation at the point of collection, enhancing research scale and diversity • Avoidance of artifacts: Minimizes stress or necrosis-related artifacts, preserving the natural cellular physiology for accurate genomic and transcriptomic analysis.<br />Competing Interests: Competing interestsH.H. is a co-founder and shareholder of Omniscope, a scientific advisory board member of MiRXES and Nanostring, and a consultant to Moderna and Singularity. L.G.M is an advisor and shareholder of Omniscope, and advisor for ArgenTAG and BioScryb. Omniscope has filed a patent related to the application of the FixNCut protocol. All other authors declare no competing interests.<br /> (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)
Details
- Language :
- English
- ISSN :
- 2331-8325
- Volume :
- 14
- Issue :
- 17
- Database :
- MEDLINE
- Journal :
- Bio-protocol
- Publication Type :
- Academic Journal
- Accession number :
- 39315321
- Full Text :
- https://doi.org/10.21769/BioProtoc.5063