Gl 7 fitc

Splenocytes and mesenteric lymph node cells were isolated and then stained with anti-mouse CD4-APC, CD8-PerCP-Cy5.5, CD19-PE, CD44-PE, CD62L-FITC, CD69-FITC, IL-7Rα-PE, CD138-APC, PSGL-1-APC, CXCR5-PE, PD-1-FITC, GL-7-FITC, CD95-PE-Cy7, B220-Alexa647 and NK1.1-FITC (eBioscience, San Diego, CA) antibodies for 15 min at 4°C. To assess intracellular cytokine levels, cells were differentiated for 5 days and then re-stimulated with Cell Stimulation Cocktail plus Protein Transport Inhibitors reagent (eBioscience) for 5 h. Then, the cells were fixed, permeabilized, and stained with anti-mouse IFN-γ-FITC, IL-4-PE, IL-17-APC, and IL-9-APC antibodies. Intracellular Foxp3 staining was performed using the Foxp3 Staining Kit (eBioscience). To determine the purity of isolated CD4⁺CD25⁻ T cells, these cells were stained with anti-CD4 and anti-CD25, and followed by FACS analysis.

All antibodies are from BD Biosciences, except where noted otherwise. CD3-biotin, CD11b-FITC -PE -PE-Cy7 -biotin, CD16/32-PE, CD19-PE, CD24-PE-Cy7, CD34-FITC, CD43-PE, CD48-FITC, CD95-PE, CD150-PE-Cy7, c-Kit-APC -FITC (eBioscience), Sca-1-PE – PE-Cy7 (eBioscience), B220-biotin, FLT3-PE (eBiosciences), Gr-1-FITC -biotin, IgM-APC, IgD-FITC (eBioscience), IL-7R-APC -biotin, TER119-biotin, GL7-FITC and TCR-APC. Cells incubated with biotinylated antibodies were treated with streptavidin conjugated with APC-Cy7 (BD Biosciences). Multipotent progenitors (MPPs) as FLT3⁺ LSK cells, common lymphoid progenitors (CLPs) as Lin⁻, IL-7R⁺ Sca-1^lo c-Kit^lo; granulocyte macrophage progenitors (GMPs) as Lin⁻, CD16/32^hi, CD34⁺, Sca1⁻, c-Kit⁺; common myeloid progenitors (CMPs) as Lin⁻, CD16/32^lo, CD34⁺, Sca1⁻, c-Kit⁺; and megakaryocyte erythroid progenitors (MEPs) as Lin⁻_, CD34⁻, CD16/32⁻, Sca-1⁻,c-Kit⁺. Pre-B, pro-B and prepro-B cells were determined by CD24 and CD43 staining of B220⁺, IgM⁻ bone marrow cells.