GSM2885051: hu_Th1.4; Homo sapiens; RNA-Seq
Mouse CD4 T-cells: Naïve CD4 T cells were isolated from spleens of 8-12 week old mice using EasySep™ Mouse CD4+ T Cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada). Following isolation, purified CD4 T cells were differentiated in vitro in 24-well plate coated with 8 μg/ml anti-mouse CD3ε  (145-2C11) and 8 μg/ml anti-mouse CD28 (37.51) for 72 h in the presence of 25 ng/ml recombinant mouse IL-12, 20 ng/ml recombinant mouse IL-2 and 1 μg/ml anti-mouse IL-4 (11B11) for Th1 differentiation, 50 ng/ml recombinant mouse IL-4, 20 ng/ml recombinant mouse IL-2, 1 μg/ml anti-mouse IL-12 and  1 μg/ml anti-mouse IFN-γ (XMG1.2) for Th2 differentiation, 50 ng/ml recombinant mouse IL-6, 5 ng/ml recombinant human TGF-β1, 1 μg/ml antimouse  IL-4 and 1 μg/ml anti-mouse IFN-γ for Th17 differentiation, and 5 ng/ml recombinant human TGF-β1 and 20 ng/ml recombinant mouse IL-2 for Treg differentiation. Neutrophils and Promyelocytes: Bone marrow cells were stained with anti-mouse surface markers PerCP-CD45 (30-F11), FITC-Ly6G (1A8), APC-CD115 (AFS98), PE-F4/80 (BM8), PE/Cy7-CD11c (N418), PE/Cy7- TER119 (TER119), PE/Cy7-CD19 (6D5), eFluor®450-CD11c (M1/70), PE-CD4 (GK1.5), PE Siglec-F (E50-2440). Neutrophils were CD45+CD11c+Ly6Ghi and promyelocytes were CD45+CD11c+Ly6Glo. Promyelocytes were further distinguished from monocytes as SSChiCD115-. Cell sorting was done by BD FACSAriaTM II (BD Biosciences, San Diego, CA). Human Cd4 T cells: Human peripheral blood mononuclear cells were obtained from healthy donors through the LJI Clinical Core in accordance with the La Jolla Institute for Allergy and Immunology Normal Blood Donor Program (VD-057). PBMCs were isolated using SepMate tubes (STEMCELL Technologies). Then, PBMCs were stained by the anti-human surface markers Alexa Fluor® 700 CD3 (UCHT1), eFluor®450 CD4 (OKT4), PE/Cy7-CD25 (BC96), PerCPCy5.5-CD45RA (HI100), Brilliant VioletTM 605-CD45RO (UCHL1), PE-CXCR3 (CEW33D), APC-CCR4 (D8SEE), FITC-CCR6 (R6H1). The CD4 T cell  subsets were sorted by BD FACSAriaTM Fusion (BD Biosciences) based on chemokine receptors expression. Live  CD3+CD4+CD25- cells were further gated using CD45RA and CD45RO  surface markers to differentiate between naïve and memory T cell subsets, respectively. Within the memory T cell subset, Th1 cells were CXCR3+CCR4-CCR6-. RNA libraries were prepared for sequencing using standard Illumina protocols
GEO Accession
GSM2885051