recombinant mouse il 21 receptor fc  (R&D Systems)

Journal: PLoS Pathogens

Article Title: Neddylation contributes to CD4 + T cell-mediated protective immunity against blood-stage Plasmodium infection

doi: 10.1371/journal.ppat.1007440

Figure Lengend Snippet: (A) Left, RNA-Seq analysis of activated CD44 hi CD4 + T cells from 3 mice at day 5 of P . yoelii 17XNL infection. Heat map showing differential expression of the signature genes related to Th1, Th2, Th17, Tfh and Treg lineages between Uba3 fl/fl and Uba3 ΔT mice. Right, validation of Bcl-6 expression by quantitative RT-PCR for activated CD4 + T cells as described above. (B) Representative counter plots and bar graphs showing the proportions and numbers of Tfh (PD-1 + CXCR5 + ) cells among activated CD4 + CD44 hi T cells in spleens of Uba3 fl/fl and Uba3 ΔT mice at days 0, 7 and 16 p.i. (n = 8–9 per group). (C) Immunoblotting for Bcl-6 in splenic CD4 + T cells from Uba3 fl/fl and Uba3 ΔT mice at days 0, 5, 7 p.i. (n = 4 per group), bar graphs showing densitometry of the bands relative to that of Uba3 fl/fl mice. (D) Intracellular staining of IL-21 in splenic CD4 + T cells from Uba3 fl/fl and Uba3 ΔT mice prior to (day 0) and at day 7 p.i. (n = 6 per group). Data are representative of two or more replicate experiments and are shown as mean±SEM. * p <0.05, *** p <0.01 by Student’s t test.

Article Snippet: IL-21 was detected using recombinant mouse IL-21 receptor/human Fc chimera (R&D Systems) and PE-conjugated anti-human IgG Fc (ebioscience), as previously described [ ].

Techniques: RNA Sequencing Assay, Infection, Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Frontiers in Immunology

Article Title: NK1.1 Expression Defines a Population of CD4 + Effector T Cells Displaying Th1 and Tfh Cell Properties That Support Early Antibody Production During Plasmodium yoelii Infection

doi: 10.3389/fimmu.2018.02277

Figure Lengend Snippet: NK1.1 + CD4 + T cells express Tbet and Bcl6 and produce effector cytokines. (A) Representative intracellular staining of NK1.1 − and NK1.1 + CD4 + T cells for the transcription factors Tbet, Bcl6, RORγt, Gata3, and Foxp3. Cells previously gated on live, CD44 hi CD62L lo CD4 + TCRβ + splenocytes. Gates based on FMO controls shown in Supplemental Figure . (B) Frequency of NK1.1 + and NK1.1 − CD4 + T cells expressing Tbet, Bcl6 or, Tbet and Bcl6 on days 8 and 11 p.i. An aligned rank transformation was performed on non-parametric data before determining significance by two-way ANOVA with a post hoc Holm-Sidak's multiple comparisons test. * p < 0.05, ** p < 0.01. (C) Median fluorescence intensity (MFI) for Tbet and Bcl6 in NK1.1 + and NK1.1 − CD4 + T cells on day 8 and 11 p.i. An aligned rank transformation was performed on non-parametric data before determining significance by two-way ANOVA with a post hoc Holm-Sidak's multiple comparisons test. * p < 0.05. (D) Representative intracellular cytokine staining of NK1.1 − and NK1.1 + CD4 + T cells previously gated on live CD4 + TCRβ + T cells, on day 7 post-infection. Splenocytes were stimulated for 4 h with PMA and ionomycin in the presence of brefeldin A before being stained for IFN-γ, IL-21, and TNFα. (E) The frequency of single, double, or triple cytokine-producing cells at day 7 post-infection. Significance assessed via an unpaired non-parametric Mann-Whitney test. * p < 0.05. Data are representative of three independent experiments (error bars, s.e.m.).

Article Snippet: For IL-21 staining, recombinant mouse IL-21 receptor fused to human Fc (R & D Systems) staining was performed first, followed by secondary anti-human Fc-PE Ab (ThermoFisher Scientific) staining.

Techniques: Staining, Expressing, Transformation Assay, Fluorescence, Infection, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: NK1.1 Expression Defines a Population of CD4 + Effector T Cells Displaying Th1 and Tfh Cell Properties That Support Early Antibody Production During Plasmodium yoelii Infection

doi: 10.3389/fimmu.2018.02277

Figure Lengend Snippet: NK1.1 + CD4 + T cells express Tbet and Bcl6 and produce effector cytokines. (A) Representative intracellular staining of NK1.1 − and NK1.1 + CD4 + T cells for the transcription factors Tbet, Bcl6, RORγt, Gata3, and Foxp3. Cells previously gated on live, CD44 hi CD62L lo CD4 + TCRβ + splenocytes. Gates based on FMO controls shown in Supplemental Figure . (B) Frequency of NK1.1 + and NK1.1 − CD4 + T cells expressing Tbet, Bcl6 or, Tbet and Bcl6 on days 8 and 11 p.i. An aligned rank transformation was performed on non-parametric data before determining significance by two-way ANOVA with a post hoc Holm-Sidak's multiple comparisons test. * p < 0.05, ** p < 0.01. (C) Median fluorescence intensity (MFI) for Tbet and Bcl6 in NK1.1 + and NK1.1 − CD4 + T cells on day 8 and 11 p.i. An aligned rank transformation was performed on non-parametric data before determining significance by two-way ANOVA with a post hoc Holm-Sidak's multiple comparisons test. * p < 0.05. (D) Representative intracellular cytokine staining of NK1.1 − and NK1.1 + CD4 + T cells previously gated on live CD4 + TCRβ + T cells, on day 7 post-infection. Splenocytes were stimulated for 4 h with PMA and ionomycin in the presence of brefeldin A before being stained for IFN-γ, IL-21, and TNFα. (E) The frequency of single, double, or triple cytokine-producing cells at day 7 post-infection. Significance assessed via an unpaired non-parametric Mann-Whitney test. * p < 0.05. Data are representative of three independent experiments (error bars, s.e.m.).

Article Snippet: For IL-21 staining, recombinant mouse IL-21 receptor fused to human Fc (R & D Systems) staining was performed first, followed by secondary anti-human Fc-PE Ab (ThermoFisher Scientific) staining.

Techniques: Staining, Expressing, Transformation Assay, Fluorescence, Infection, MANN-WHITNEY