Related Experiment Video
Updated: Jul 24, 2025
Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing
Published on: December 3, 2019
Optimized protocol for mouse footpad immune cell isolation for single-cell RNA sequencing and flow cytometry
Muddassar Hameed1, Pallavi Rai1, Melissa Makris2
1Department of Biomedical Sciences and Pathobiology, VA-MD Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24060, USA; Center for Zoonotic and Arthropod-borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA.
This study details a method for isolating mouse footpad leukocytes with high viability for single-cell RNA sequencing (scRNA-seq). This protocol enables the creation of a comprehensive single-cell molecular atlas.
Area of Science:
- Immunology
- Genomics
- Biotechnology
Background:
- High cell viability is crucial for reliable single-cell RNA sequencing (scRNA-seq) results.
- Isolating leukocytes from challenging tissues like the mouse footpad requires optimized protocols.
Purpose of the Study:
- To present a detailed protocol for isolating mouse footpad leukocytes.
- To ensure high cell viability during the isolation process for downstream scRNA-seq applications.
Main Methods:
- Footpad tissue collection and enzymatic dissociation.
- Leukocyte isolation, purification, fixation, and preservation.
- Combinatorial barcoding, library preparation, scRNA-seq, and data analysis.
Main Results:
- Successful isolation of mouse footpad leukocytes with high viability.
- Demonstration of the protocol's suitability for generating a single-cell molecular atlas.
Conclusions:
- The presented protocol effectively isolates viable leukocytes from mouse footpads.
- This method facilitates comprehensive single-cell analysis and the construction of molecular atlases.
