Retina and secondary lymphoid organs (SLO, including draining LN and spleens) from CAU mice (week 12 – 16 post-induction) were harvested and pooled, and CD4+ T cells were isolated via negative selection with a magnetic CD4+ T cell isolation kit (Cat #: 130-104-454, Miltenyi Biotec Inc.). The isolated cells were >98% CD4+ as confirmed by flow cytometry, and they were stained with FITC- or PerCP/Cy5.5-anti-CD44 (clone IM7, Cat #: 103006 or 103032, BioLegend) antibody, and sorted for CD44hi memory and CD44−~lo control subpopulations using a MoFlo® FACS sorter (Dako Cytomation) or a BD FACSAria™ III sorter (BD Biosciences). The sorted cells in equal live numbers (1×105) were cultured for 5 days and stained with 7-AAD (Cat #: 00-6993-50, ThermoFisher), and the viable cells were determined as 7-AAD unstained populations by flow cytometric analysis.
Fitc or percp cy5.5 anti cd44 clone im7
Manufactured by BioLegend
FITC- or PerCP/Cy5.5-anti-CD44 (clone IM7) is a fluorescently labeled antibody that binds to the CD44 cell surface antigen. CD44 is a cell adhesion molecule involved in various cellular processes. This product can be used to detect and analyze CD44-expressing cells in flow cytometry applications.
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2 protocols using fitc or percp cy5.5 anti cd44 clone im7
1
CD4+ T Cell Subset Isolation
2
Isolation and Sorting of CD4+ T Cell Subsets
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Retina and secondary lymphoid organs (SLO, including draining LN and spleens) from CAU mice (week 12 -16 post-induction) were harvested and pooled, and CD4 + T cells were isolated via negative selection with a magnetic CD4 + T cell isolation kit (Cat #: 130-104-454, Miltenyi Biotec Inc.). The isolated cells were >98% CD4 + as confirmed by flow cytometry, and they were stained with FITC-or PerCP/Cy5.5-anti-CD44 (clone IM7, Cat #: 103006 or 103032, BioLegend) antibody, and sorted for CD44 hi memory and CD44 -~lo control subpopulations using a MoFlo ® FACS sorter (Dako Cytomation) or a BD FACSAria ™ III sorter (BD Biosciences). The sorted cells in equal live numbers (1×10 5 ) were cultured for 5 days and stained with 7-AAD (Cat #: 00-6993-50, ThermoFisher), and the viable cells were determined as 7-AAD unstained populations by flow cytometric analysis.
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