Bv605 anti mouse cd4

APC anti-mouse CD4 (clone: RM4-5), BV605 anti-mouse CD4 (clone: GK1.5), FITC anti-mouse CD3ε, PE anti-mouse CD8α, V500 rat anti-mouse B220, FITC anti-mouse CD45R/B220, APC anti-mouse IgD, APC anti-mouse CD21/CD35, PE anti-mouse CD23, FITC anti-mouse CD8, BV786 anti-mouse CD3, PE CF594 anti-mouse CD23, APC Cy7 anti-mouse CD21, FITC anti-mouse Gr1, APC anti-mouse CD11b, PE anti-mouse F4/80, FITC anti-mouse I-A/E, and APC streptavidin were bought from BD bioscience (San Jose, CA). Biotinylated anti-mouse C3 was obtained from Cedarlane (Burlington, Canada), and APC anti-mouse IgG, APC anti-mouse IgM and FITC anti-mouse IgM were purchased from Jackson Immunoresearch (West Grove, PA). PE anti-mouse CD1d and Zombie yellow live dead was purchased from Biolegend (San diego, CA).

Colon lamina propria cells were subsequently counted, and the surface of the cells were stained for 30 min at room temperature using Fixable viability stain 510 (564406, BD Biosciences) for live cell staining, Alexa Fluor 700 anti-mouse CD45 (560510, BD Biosciences), PECP-CY5.5 anti-mouse CD3 (551163, BD Biosciences) and BV605 anti-mouse CD4 (563151, BD Biosciences) for cell surface staining. Next, cells were permeabilized with fixation/permeabilization buffer (eBioscience, San Diego, CA, USA) and cells were stained for 40 min at room temperature using PE-CY7 anti-mouse foxp3(560408, BD Biosciences), BV421 anti-mouse gata3 (563349, BD Biosciences), AF647 anti-mouse T-bet (561267, BD Biosciences), PE anti-mouse RORγt (IC6006P-025, R&D), APC anti-mouse helios (137222, Biolegend) for intracellular staining. Samples were analyzed using Flow Cytometer (BD Biosciences). Subsequent analysis was performed with FlowJo software (Tree Star Inc., San Carlos, CA). CD4 T cells were identified as CD45⁺CD3⁺CD4⁺; Treg cells were identified as CD45⁺CD3⁺CD4⁺foxp3⁺; Th2 cells were identified as CD45⁺CD3⁺CD4⁺gata3⁺foxp3⁻; Th1 cells were identified as CD45⁺CD3⁺CD4⁺T-bet⁺; Th17 cells were identified as CD45⁺CD3⁺CD4⁺RORγt ⁺foxp3⁻. nTreg were identified as CD45⁺CD3⁺CD4⁺Foxp3⁺helios⁺.