Jul 16, 2023
FACS for CD45+ immune cells
- 1Columbia University
Protocol Citation: Connor Monahan 2023. FACS for CD45+ immune cells. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3465lk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 12, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84890
Keywords: FACS, immune cell, T cell, sorting, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 0375
Abstract
This protocol details FACS of intestinal immune cells (CD45+).
Attachments
786-1993.pdf
45KB
Materials
Reagents
BD Pharmingen™ Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™)BD BiosciencesCatalog #553142
BD Horizon™ BUV395 Rat Anti-Mouse CD45BD BiosciencesCatalog #564279
Propidium Iodide SolutionBioLegendCatalog #421301
Equipment
Sony MA900 Multi-Application Cell Sorter
Procedure
Procedure
52m
52m
Resuspended cells isolated from the small intestine with 400 µL Fc block (BD Biosciences,
Cat# 553142; Franklin Lakes, NJ), 1:200 in FACS buffer.
Incubate for 00:20:00 On ice .
20m
Wash cells in 500 µL FACS buffer, pellet at 1500 rpm, 00:02:00 , then resuspend in 600 µL of FACS buffer with 1:100 CD45 BUV395 antibody (BD Biosciences, Cat# 564279; Franklin Lakes, NJ).
2m
Incubate On ice for 00:30:00 .
30m
After 30 minutes, wash cells in 500 µL FACS buffer.
Resuspend in 500 µL of FACS buffer containing propidium iodide (BioLegend, Cat# 421301; San Diego, CA) at 1:10,000.
Sort viable, CD45+ cells on a Sony MA900 Multi-Application Cell Sorter.