dc t cell co cultures cd4 t cells  (Miltenyi Biotec)

Journal: Cellular and Molecular Immunology

Article Title: Deficiency of the G protein Gαq ameliorates experimental autoimmune encephalomyelitis with impaired DC-derived IL-6 production and Th17 differentiation

doi: 10.1038/cmi.2016.65

Figure Lengend Snippet: Gαq-KO inhibits in vivo Th17 development. Leukocytes were isolated from the lymph nodes of WT mice and Gαq-KO mice on day 10 PI and analyzed with flow cytometry. (a–c) Th1 and Th17 cells were analyzed by intracellular staining of IFN-γ and IL-17A, respectively, in the CD4+ gate. (d–f) Lymph node leukocytes from EAE-induced WT mice and Gαq-KO mice were re-stimulated in vitro with MOG35–55 (20 μg/ml) for 72 h, and IL-17A (d), IFN-γ (e) and TGF-β (f) in supernatants were analyzed with ELISA. (g) qPCR analysis of Th1- and Th17-related gene expression in lymph nodes. The data are expressed as the mean±s.e.m. (n=3), * P<0.05 versus WT-EAE.

Article Snippet: DC-T cell co-culture A CD4 + CD62L + T cell Isolation Kit II (Miltenyi Biotec, Auburn, CA, USA) was used to isolate naive T cells from single-cell suspensions from the spleen; in the first step, non-CD4 + cells were magnetically depleted, and magnetically labeled CD62L + T cells were positively selected in the second step.

Techniques: In Vivo, Isolation, Flow Cytometry, Staining, In Vitro, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: Cellular and Molecular Immunology

Article Title: Deficiency of the G protein Gαq ameliorates experimental autoimmune encephalomyelitis with impaired DC-derived IL-6 production and Th17 differentiation

doi: 10.1038/cmi.2016.65

Figure Lengend Snippet: Gαq-KO inhibits in vitro Th17 differentiation. (a–f) Naive CD4+ T cells from WT and Gαq-KO mice were induced to differentiate into Th1 (a, b) or Th17 (d, e) cells in vitro. Antigen-specific Th1 and Th17 responses were measured by ELISA for IFN-γ and IL-17A production in the supernatant (c, f). The data are expressed as the mean±s.e.m. (n=3), ***P<0.001 versus WT.

Article Snippet: DC-T cell co-culture A CD4 + CD62L + T cell Isolation Kit II (Miltenyi Biotec, Auburn, CA, USA) was used to isolate naive T cells from single-cell suspensions from the spleen; in the first step, non-CD4 + cells were magnetically depleted, and magnetically labeled CD62L + T cells were positively selected in the second step.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Human Vaccines & Immunotherapeutics

Article Title: Thimerosal compromises human dendritic cell maturation, IL-12 production, chemokine release, and T-helper polarization

doi: 10.4161/hv.29520

Figure Lengend Snippet: Figure 1. Comparative susceptibility of primary T cells and monocyte-derived DCs to thimerosal toxicity. PBMC from a HD were incubated overnight in medium (unstimulated) (A) or stimulated with anti-CD3/CD28 mAbs (B) in the presence of thimerosal at indicated concentrations. Cell viability was analyzed by flow cytometry, combining CD4 staining with 7-AAD to quantify apoptosis. (C): monocyte-derived DCs were prepared as described in Material and Methods, and matured for 24h with LPS (10 µg/mL) in the presence of various concentrations of thimerosal. The level of cell death in LPS-matured DCs was determined combining CD86 and 7-AAD staining. These dot plots are representative of 3 independent experiments.

Article Snippet: DC – T CD4 + cocultures Naïve CD3 + CD4 + T cells (CD3 + CD4 + CD45RA + ) were sorted from PBMCs by negative selection using immunomagnetic beads (Miltenyi Biotech).

Techniques: Derivative Assay, Incubation, Flow Cytometry, Staining

Journal: Human Vaccines & Immunotherapeutics

Article Title: Thimerosal compromises human dendritic cell maturation, IL-12 production, chemokine release, and T-helper polarization

doi: 10.4161/hv.29520

Figure Lengend Snippet: Figure 5. Impact of thimerosal on Th polarization by mature DCs. Pattern of cytokines and chemokines simultaneously quantified by multiplex bead assay arrays in supernatants from LPS-matured DCs (48 stimulation of iDCs with LPS 10 µg/mL) co-cultured for 5 d with sorted allogeneic or syngeneic naïve CD4+ T cells in the presence of 2 different concentrations of thimerosal (0.18 and 0.36 µg/mL). (A) Heat map of the percentage of inhibition or activation of cytokine/chemokine expression in the presence of thimerosal. Data are representative of 3 experiments. (B) Representative data of the pattern of cytokines and chemokines that were inhibited or increased by thimerosal.

Article Snippet: DC – T CD4 + cocultures Naïve CD3 + CD4 + T cells (CD3 + CD4 + CD45RA + ) were sorted from PBMCs by negative selection using immunomagnetic beads (Miltenyi Biotech).

Techniques: Multiplex Assay, Cell Culture, Inhibition, Activation Assay, Expressing