The phenotypes of DC as well as the proliferation assay of CFSE-labeled T cells were determined by flow cytometry35 (link). For cell surface staining, single-cell suspensions were incubated for 15 min at 4 °C with PE Hamster anti-mouse CD11c (0.4 μg/ml) (BD Biosciences, 553802), PerCP-Cy5.5 Hamster anti-mouse CD80 (0.2 μg/ml) (BD Biosciences, 560526), PerCP-Cy5.5 anti-mouse CD86 (0.2 μg/ml) (Biolegend, 105027), PE-Cy7 anti-mouse CD40 (0.1 μg/ml) (Biolegend, 124621), PE-Cy7 anti-mouse I-Ab (0.1 μg/ml) (Biolegend, 116420), PE-Cy7 anti-mouse CD4 (0.2 μg/ml) (Biolegend, 100422), APC anti-mouse TCR Vβ5.1 antibody (0.2 μg/ml) (Biolegend, 139511), and FITC anti-mouse CD45.2 antibody (0.2 μg/ml) (Biolegend, 109805). Samples were washed and analyzed by FACS versus flow cytometry (BD Biosciences), and the gating strategies were shown in Supplementary Fig. 9 .
Fitc anti mouse cd45.2 antibody
Manufactured by BioLegend
The FITC anti-mouse CD45.2 antibody is a fluorescently-labeled monoclonal antibody that specifically binds to the CD45.2 isoform of the mouse CD45 protein. CD45 is a transmembrane protein tyrosine phosphatase expressed on the surface of most hematopoietic cells.
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2 protocols using fitc anti mouse cd45.2 antibody
1
Phenotype and Proliferation Analysis of Dendritic Cells and T Cells
2
Determining Donor Cell Engraftment Efficiency
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CD45 is a type 1 transmembrane protein that is present on all differentiated hematopoietic cells and is a useful marker for determining bone marrow transplant efficiency (Omilusik et al., 2011 (link)). C57Bl/6 mice are genetically CD45.2, whereas SJL mice are CD45.1, so all AD and WT littermates were tested for CD45 alleles and only those that were CD45.1 were used as donors; all APP KO recipients were CD45.2 (on C57Bl/6 background). Briefly, 50–100 μL of blood was collected into EDTA-coated tubes and lymphocytes were isolated after red blood cell (RBC) lysis. Isolated lymphocytes were then incubated with antibodies against CD45.2 (FITC anti-mouse CD45.2 antibody; BioLegend; 109805) and CD45.1(PE/Cy7 anti-mouse CD45.1 Antibody (Biolegend; 110729) in FACS buffer (phosphate-buffered saline [PBS], 0.5%–1% BSA or 5%–10% FBS). The samples were analyzed for CD45 type using a BD LSRII (BD Biosciences). Lymphocytes were gated based on size and shape, and gating was consistent among all samples. The representative graph was normalized to mode, which allows the visualization of the relative percentage of a cell population expressing the marker fluorescence and compensates for the visual difference in cell count. FlowJo software (Treestar) was used to analyze flow cytometry data.
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