Alexa fluor 488 anti mouse rat human foxp3 antibody

Brilliant Violet 421™ anti-mouse I-A/I-E Antibody, Brilliant Violet 510™ anti-mouse CD4 Antibody, Brilliant Violet 570™ anti-mouse/human CD45R/B220 antibody, Brilliant Violet 605™ anti-mouse IgM Antibody, Brilliant Violet 650™ anti-mouse CD25 antibody, Brilliant Violet 785™ anti-mouse CD8a antibody, PE/Cy7 Goat anti-mouse IgG (minimal x-reactivity) antibody, Alexa Fluor^® 488 anti-mouse/rat/human FOXP3 antibody, Alexa Fluor^® 647 anti-mouse IgD antibody, and APC/Cy7 anti-mouse CD138 (Syndecan-1) antibody were purchased from Biolegend. AnaSpec 7-AAD was procured from Fisher Scientific and Biotinylated Peanut Agglutinin (PNA), was purchased from Vector Labs. Biotinylated goat anti-mouse IgG, IgG1, IgA and IgM secondary Abs were purchased from Jackson ImmunoResearch Laboratories. For monitoring protein expression on arrays, monoclonal mouse anti-polyhistidine was procured from Sigma-Aldrich (St. Louis, MO, USA) and rat anti-hemagglutinin (HA; clone 3F10, anti-HA high affinity), from Roche (Pleasanton, CA) were used. Streptavidin-conjugated SureLight P3 was purchased from Columbia Biosciences (Frederick, MD).

FACS analysis was carried out on the blood and tissue samples on a Beckman Coulter Gallios Flow Cytometer. Antibodies specific for the following cell surface markers were used: CD3 conjugated to PerCP-Cy5.5 (clone: 145-2C11), CD4 conjugated to PE (clone: GK1.5), CD8 conjugated to BV421 (clone: 53 − 6.7), and CD25 conjugated to APC (clone: PC61). Moreover, the measurement of intracellular signaling molecules (Alexa Fluor® 488 anti-mouse/rat/human FOXP3 antibody, clone: 150D, BioLegend, USA) was performed for Foxp3 analysis. Each experiment included single-color compensation and an appropriate isotype control, and each sample was independently analyzed at least three times. As evaluated by the mean fluorescence intensity, the percentage of positive cells was determined with FlowJo software (Tree Star, Inc. OR, USA). Lymphocytes were identified by plotting forward vs. side scatter followed by gating on CD3 + T cells, and these cells were then analyzed by separation into CD4 + T and CD8 + T cells. Treg cells were identified as CD25 + Foxp3 + cells gated within CD3 + CD4 + T cells.