immunomagnetic negative selection easysep mouse t cell isolation kit  (STEMCELL Technologies Inc)

Journal: JCI Insight

Article Title: Histone demethylase JARID1C/KDM5C regulates Th17 cells by increasing IL-6 expression in diabetic plasmacytoid dendritic cells

doi: 10.1172/jci.insight.172959

Figure Lengend Snippet: ( A ) Representative flow cytometry gating schematic to isolate pDCs (CD45 + , Ly6C + , CD11c + , PDCA1 + ) from murine diabetic wounds. ( B ) Kinetic plot of diabetic wound pDCs from 6 mm wounds over time ( N = 5/group, pooled and repeated in triplicate) determined by flow cytometry. ( C ) mRNA fold expression of Il6 and Tgfb in isolated wound pDCs from diabetic and nondiabetic control mice. ( N = 3–5/group, pooled, repeated in triplicate.) ( D ) Protein expression of IL-6 and TGF-β in isolated wound pDCs from diabetic and nondiabetic control mice. ( N = 3–5/group, pooled, repeated in triplicate.) Wound pDCs were isolated using EasySep magnetic bead pDC negative-selection kit, according to manufacturer instructions. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are presented as the mean ± SEM and were analyzed using 2-tailed Student’s t test.

Article Snippet: The spleens were crushed and filtered through a 100-micron filter, and the suspension was subjected to magnetic activated cell sorting (MACS) using a negative-selection EasySep Mouse CD4 + naive T cell isolation kit (STEMCELL Technologies) according to manufacturer instructions.

Techniques: Flow Cytometry, Expressing, Isolation, Control, Selection

Journal: JCI Insight

Article Title: Histone demethylase JARID1C/KDM5C regulates Th17 cells by increasing IL-6 expression in diabetic plasmacytoid dendritic cells

doi: 10.1172/jci.insight.172959

Figure Lengend Snippet: ( A ) Jarid1c expression in ND and DIO wound pDCs. ( N = 6/group, pooled, repeated in triplicate.) ( B ) ChIP of the H3K4me3 mark on the Il6 promoter in wound pDCs. ( N = 6/group, pooled, repeated in triplicate.) ( C ) ChIP of Jarid1c at the Il6 promoter in wound pDCs. ( N = 3–5/group, pooled, repeated in triplicate.) ( D ) Fold expression of Il6 in diabetic wound pDCs with and without recombinant JARID1C (rJARID1C, 10 nM, 24 hours; N = 3–5/group, pooled, repeated in triplicate). ( E ) Protein expression determined by ELISA of IL-6 in diabetic wound pDCs with and without rJARID1C (10 nM, 24 hours; N = 3–5/group, pooled, repeated in triplicate). ( F ) Fold expression of stimulated CD4 + T cell Il17a following incubation with supernatant from nondiabetic wound pDCs with and without JARID1 inhibition for 24 hours (KDOAM25, 500 nM; N = 3/group, pooled, repeated in triplicate) in the presence and absence of anti–IL-6 antibody. For each experiment, wound pDCs were harvested on day 1 after injury and isolated using EasySep pDC negative-selection magnetic bead kit. * P < 0.05, ** P < 0.01. All data are presented as mean ± SEM. Data in F were statistically analyzed using 1-way ANOVA with Holm-Šidák multiple-comparison test. For all other panels, data were analyzed using 2-tailed Student’s t test once normality was assessed.

Article Snippet: The spleens were crushed and filtered through a 100-micron filter, and the suspension was subjected to magnetic activated cell sorting (MACS) using a negative-selection EasySep Mouse CD4 + naive T cell isolation kit (STEMCELL Technologies) according to manufacturer instructions.

Techniques: Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation, Inhibition, Isolation, Selection, Comparison

Journal: JCI Insight

Article Title: Histone demethylase JARID1C/KDM5C regulates Th17 cells by increasing IL-6 expression in diabetic plasmacytoid dendritic cells

doi: 10.1172/jci.insight.172959

Figure Lengend Snippet: ( A ) Il6 expression in DIO wound pDCs with and without recombinant IFN-β. ( B ) ChIP analysis of Jarid1c on the Il6 promoter in diabetic wound pDCs with and without IFN-β stimulation. ( C ) ChIP of the H3K4me3 mark on the Il6 promoter in DIO wound pDCs with and without IFN-β stimulation. ( D ) Il6 expression in DIO wound pDCs with IFN-β stimulation only with and without JARID1 inhibition. ( E ) ChIP of the H3K4me3 mark on the Il6 promoter in DIO wound pDCs with IFN-β stimulation, with and without JARID1 inhibition. The graphs are color-coded according to the key, which denotes the cell treatment. ( F ) ChIP of Jarid1c at the Il6 promoter in IFNAR –/– mouse wound pDCs compared with their age-matched littermate controls. ( G ) ChIP of the H3K4me3 mark on the Il6 promoter in IFNAR –/– mouse wound pDCs, compared with their age-matched littermate controls. ( H ) Il6 expression in wound pDCs from IFNAR –/– mice and their age-matched littermate controls. ( I ) ChIP of Jarid1c at the Il6 promoter in DIO mouse wound pDCs with a TYK2 inhibitor. ( J ) ChIP of the H3K4me3 mark at the Il6 promoter in DIO mouse wound pDCs with TYK2 inhibition. ( K ) Il6 expression in DIO wound pDCs following TYK2 inhibition. ( L ) ChIP of Jarid1c on the Il6 promoter in DIO wound PDCs following JAK1,3 inhibition. ( M ) ChIP of the H3K4me3 mark on the Il6 promoter in DIO mouse wound pDCs following JAK1,3 inhibition. ( N ) Il6 expression in DIO wound pDCs after JAK1,3 inhibition. * P < 0.05, ** P < 0.01, *** P < 0.001. All experiments were conducted with N = 3–6 mice/group, pooled and repeated in triplicate. Murine wound pDCs were harvested on day 1 after injury and isolated using EasySep pDC negative-selection kit. Data are presented as the mean ± SEM. Data were first analyzed for normal distribution, and if data passed the normality test, 2-tailed Student’s t test was used.

Article Snippet: The spleens were crushed and filtered through a 100-micron filter, and the suspension was subjected to magnetic activated cell sorting (MACS) using a negative-selection EasySep Mouse CD4 + naive T cell isolation kit (STEMCELL Technologies) according to manufacturer instructions.

Techniques: Expressing, Recombinant, Inhibition, Isolation, Selection

Journal: JCI Insight

Article Title: Histone demethylase JARID1C/KDM5C regulates Th17 cells by increasing IL-6 expression in diabetic plasmacytoid dendritic cells

doi: 10.1172/jci.insight.172959

Figure Lengend Snippet: ( A ) Flow cytometry analysis of RORγt expression in naive CD4 + T cells cocultured for 48–72 hours with DIO and ND wound pDCs. ( B ) Flow cytometry analysis of IL-17A in CD4 + T cells cocultured for 48–72 hours with DIO and ND wound pDCs. ( C ) RORγt expression in CD4 + T cells after 48- to 72-hour coculture with DIO wound pDCs under several conditions — naive CD4 + T cells (control), naive CD4 + T cells with IL-6 preinhibition (LMT-28, 200 nM, 1 hour), TGF-β receptor–deficient naive CD4 + T cells (Alk5 fl/fl CD4 Cre+ T cells), and Alk5 fl/fl CD4 Cre+ CD4 + T cells with IL-6 receptor preinhibition (LMT-28, 200 nM, 1 hour). ( D ) IL-17A expression in CD4 + T cells after 48- to 72-hour coculture with DIO wound pDCs under various conditions, as detailed in C . For these coculture experiments, each mouse received 3–4 wounds, and N = 3–5 mice/group, pooled and repeated in triplicate. ( E ) Cluster uniform manifold approximation and projection (UMAP) of scRNA-Seq from human T2D and non-T2D wounds showed 10 unique cell clusters (representative). ( F ) scRNA-Seq of human wound T-cell population demonstrating RORγt expression in T2D versus non-T2D controls ( N = 42). Dot size corresponds to proportion of cells within the group expressing RORγt, while dot color corresponds to expression level. ( G ) Intracellular flow cytometry quantifying intracellular RORγt in ND and DIO wound CD4 + T cells. ( N = 3–5 mice/group, pooled and repeated in triplicate.) ( H ) Protein expression by ELISA of IL-17A in ND versus DIO wound CD4 + T cells ( N = 5/group, pooled and repeated in triplicate; day 5 wounds). ( I ) Wound healing curve in global knockout, diabetic IL17A –/– mice compared with age-matched, littermate controls ( N = 3/group, pooled and repeated in triplicate). ( J ) Representative wound healing images and histology. Data are presented as the mean ± SEM. For C and D , data were analyzed using 1-way ANOVA with Holm-Šídák multiple-comparison test. Data for I were analyzed using 2-way repeated measures ANOVA. For all other panels, data were analyzed using 2-tailed Student’s t test once normality was assessed.

Article Snippet: The spleens were crushed and filtered through a 100-micron filter, and the suspension was subjected to magnetic activated cell sorting (MACS) using a negative-selection EasySep Mouse CD4 + naive T cell isolation kit (STEMCELL Technologies) according to manufacturer instructions.

Techniques: Flow Cytometry, Expressing, Control, Enzyme-linked Immunosorbent Assay, Knock-Out, Comparison