FACS for CD45+ immune cells
Connor Monahan
Published: 2023-07-17 DOI: 10.17504/protocols.io.36wgq3465lk5/v1
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Abstract
This protocol details FACS of intestinal immune cells (CD45+).
Attachments
786-1993.pdf
Steps
Procedure
1.
Resuspended cells isolated from the small intestine with 400µL Fc block (BD Biosciences,
Cat# 553142; Franklin Lakes, NJ), 1:200 in FACS buffer.
2.
Incubate for 0h 20m 0s On ice.
3.
Wash cells in 500µL FACS buffer, pellet at 1500rpm, then resuspend in 600µL of FACS buffer with 1:100 CD45 BUV395 antibody (BD Biosciences, Cat# 564279; Franklin Lakes, NJ).
4.
Incubate On ice for 0h 30m 0s.
5.
After 30 minutes, wash cells in 500µL FACS buffer.
6.
Resuspend in 500µL of FACS buffer containing propidium iodide (BioLegend, Cat# 421301; San Diego, CA) at 1:10,000.
7.
Sort viable, CD45+ cells on a Sony MA900 Multi-Application Cell Sorter.
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