easysep human memory cd4+ t cell enrichment kit  (STEMCELL Technologies Inc)

Journal: Frontiers in Immunology

Article Title: The β 2 -adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner

doi: 10.3389/fimmu.2024.1446424

Figure Lengend Snippet: Intracellular cytokine staining and expression of transcription factor genes associated with Th1, Th2, and Th17 in memory Th cells treated with nebivolol. Memory Th cells were not activated or activated with anti-CD3/anti-CD28/anti-CD2 for 5 days, and the samples were stained for intracellular cytokines with or without nebivolol treatment and analyzed by flow cytometry. Representative dot plots are shown for CD4 and each of IFN-γ, IL-4, and IL-17A antibodies on memory Th lymphocytes. (A) Non-activated cells, (B) activated cells, (C) activated cells plus nebivolol, (D) vehicle control. (E) The proportion of memory Th cells expressing IFN-γ is shown as the percentage of IFN-γ + CD4 + T cells. (F) The proportion of CD4 + T cells expressing IL-4 is shown as the percentage of IL-4 + CD4 + T cells. (G) The proportion of CD4 + T cells expressing IL-17A is shown as the percentage of IL-17A + CD4 + T cells. Expression of (H) TBX21 , (I) GATA3 , and (J) RORC in RNA extracted from memory Th cells, shown as the relative amounts normalized to housekeeping RNA and compared to the Act group, which was set to 1.0 (dotted line). Pooled data are expressed as mean ± SEM of five independent biological experiments. One-way ANOVA followed by Tukey’s multiple comparison tests. *<0.05, **<0.01, and ****p<0.0001. ns, non-significant.

Article Snippet: Naive (CD3 + CD4 + CD45RA + CD45RO − ) and memory (CD3 + CD4 + CD45RA − CD45RO + ) T cells were isolated from peripheral blood mononuclear cells (PBMCs) in the recommended medium (PBS containing 2% FBS and 1 mM of EDTA) by using EasySep ® Human Naive CD4 + T Cell Isolation Kit II and human memory CD4 + T-cell enrichment kit, respectively (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions.

Techniques: Staining, Expressing, Flow Cytometry, Control, Comparison

Journal: Frontiers in Immunology

Article Title: The β 2 -adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner

doi: 10.3389/fimmu.2024.1446424

Figure Lengend Snippet: Nebivolol inhibits naive Th cell shift toward the Th17 phenotype, in contrast to terbutaline which augments the shift. (A) Expression of ADRB1 –3 in RNA extracted from naive CD4 + T cells shown as the relative amounts normalized to housekeeping RNA. (B) Naive Th cells were activated with polarizing cytokines and blocking antibodies (Th17) with treatment with nebivolol (Neb) or terbutaline (Terb) for 7 days, and a representative of IL-17A levels in cell culture supernatants was shown. (C) RNA expression of the Th17 cell-specific transcriptional factor RORC differentiated Th17 cells shown as the relative amounts normalized to housekeeping RNA and compared to shifted Th17 cells, which was set to 1.0 (dotted line). (D) A representative of overlapping histogram and (E) pooled data of intracellular cytokine staining shown for CD4 + IL-17A + cell percentage in polarized Th17 cells after treatment with nebivolol or terbutaline. Pooled data are expressed as mean ± SEM of five independent biological experiments. One-way ANOVA followed by Tukey’s multiple comparison tests. *<0.05, **<0.01, ***p<0.001, and ****p<0.0001.

Article Snippet: Naive (CD3 + CD4 + CD45RA + CD45RO − ) and memory (CD3 + CD4 + CD45RA − CD45RO + ) T cells were isolated from peripheral blood mononuclear cells (PBMCs) in the recommended medium (PBS containing 2% FBS and 1 mM of EDTA) by using EasySep ® Human Naive CD4 + T Cell Isolation Kit II and human memory CD4 + T-cell enrichment kit, respectively (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions.

Techniques: Expressing, Blocking Assay, Cell Culture, RNA Expression, Staining, Comparison

Journal: Frontiers in Immunology

Article Title: The β 2 -adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner

doi: 10.3389/fimmu.2024.1446424

Figure Lengend Snippet: T-cell proliferation and viability were measured by flow cytometry assay. Memory Th cells were not activated or activated with anti-CD3/anti-CD28/anti-CD2 for 5 days. Representative histograms of the proliferation of memory Th cells in cultures with different groups including (A) activated cells overlayed with non-activated cells, (B) activated cells plus nebivolol overlayed with activation-only condition, and (C) activated vehicle control overlayed with activation-only cells. (D) The proliferation index of memory Th cells in culture with activation and nebivolol. (E) The division index of pooled data. (F) The percentage of alive memory CD4 + , 7AAD − T cells with or without treatment for 5 days gated on all cells in a dot plot by staining with 7AAD in cultures with different groups. Pooled data are expressed as mean ± SEM of four independent biological experiments. One-way ANOVA followed by Tukey’s multiple comparison tests. ****p<0.0001. ns, non-significant.

Article Snippet: Naive (CD3 + CD4 + CD45RA + CD45RO − ) and memory (CD3 + CD4 + CD45RA − CD45RO + ) T cells were isolated from peripheral blood mononuclear cells (PBMCs) in the recommended medium (PBS containing 2% FBS and 1 mM of EDTA) by using EasySep ® Human Naive CD4 + T Cell Isolation Kit II and human memory CD4 + T-cell enrichment kit, respectively (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Activation Assay, Control, Staining, Comparison