th17 medium  (Proteintech)

Journal: The Journal of Clinical Investigation

Article Title: A20’s linear ubiquitin–binding motif restrains pathogenic activation of Th17 cells and IL-22–driven enteritis

doi: 10.1172/JCI187499

Figure Lengend Snippet: ( A ) Gene ontology enrichment of genes that are significantly upregulated in A20 ZF7 versus WT intestines by bulk RNA-Seq. Red bars highlight categories related to Th17 differentiation. Blue bars highlight categories related to cellular proliferation. ( B ) qPCR analyses of mRNA expression from intact small intestine. ( C ) Volcano plot of all annotated UCSC RefSeq genes from bulk RNA-Seq analyses of A20 ZF7 versus WT small intestines. Horizontal dotted line indicates adjusted P value (FDR) of 0.01. ( D ) Representative immunohistochemical analyses of CD4 expression in WT and A20 ZF7 small intestines. Data are representative of 3 mice from each genotype. Scale bars: 100 μm. ( E ) Flow cytometry of small intestinal lamina propria (SILP) cells from WT and A20 ZF7 mice. SILP yields from the proximal 10 cm of small intestine typically range from 1 million to 2 million cells from WT mice and 3 million to 7 million cells from A20 ZF7 mice. Data are shown as mean ± SEM. Statistics were calculated using unpaired 2-tailed Student’s t test with Welch’s correction. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were subsequently differentiated into Th17 cells in plates precoated with 5 μg/mL anti-CD3 (Bio X Cell BE0001-2, clone OKT3) in Th17 medium (Immunocult XF serum-free medium with 50 μM β-mercaptoethanol, 30 ng/mL recombinant human IL-6 [Proteintech HZ-1019], 2.5 ng/mL recombinant human TGF-β1 [PeproTech 100-21], 10 ng/mL recombinant human IL-1β [Proteintech HZ-1164], 10 ng/mL recombinant human IL-23 [Proteintech HZ-1254], 10 μg/mL anti–IL-4 [Bio X Cell BE0240, clone MP4-25D2], and 10 μg/mL anti–IFN-γ [Bio X Cell BE0235, clone B133.5]) at a density of 1 million cells/mL.

Techniques: RNA Sequencing, Expressing, Immunohistochemical staining, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: A20’s linear ubiquitin–binding motif restrains pathogenic activation of Th17 cells and IL-22–driven enteritis

doi: 10.1172/JCI187499

Figure Lengend Snippet: ( A and B ) UMAP clusters of scRNA-Seq analyses of SILP from WT and A20 ZF7 mice. Data represent pooled mixtures of 2 WT and 2 A20 ZF7 mice. ( C ) Relative proportions of lymphoid subsets in WT and A20 ZF7 SILP. Th17 subset includes both proliferative (mustard yellow) and non-proliferative (sky blue) compartments. ( D ) Projection of Il17a and Il22 expression onto UMAP clusters shown in B . ( E ) Violin plots of Il17a and Il22 expression in Th17 cells from indicated genotypes of mice. Statistics were calculated using unpaired 2-tailed Wilcoxon’s rank sum test. ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were subsequently differentiated into Th17 cells in plates precoated with 5 μg/mL anti-CD3 (Bio X Cell BE0001-2, clone OKT3) in Th17 medium (Immunocult XF serum-free medium with 50 μM β-mercaptoethanol, 30 ng/mL recombinant human IL-6 [Proteintech HZ-1019], 2.5 ng/mL recombinant human TGF-β1 [PeproTech 100-21], 10 ng/mL recombinant human IL-1β [Proteintech HZ-1164], 10 ng/mL recombinant human IL-23 [Proteintech HZ-1254], 10 μg/mL anti–IL-4 [Bio X Cell BE0240, clone MP4-25D2], and 10 μg/mL anti–IFN-γ [Bio X Cell BE0235, clone B133.5]) at a density of 1 million cells/mL.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: A20’s linear ubiquitin–binding motif restrains pathogenic activation of Th17 cells and IL-22–driven enteritis

doi: 10.1172/JCI187499

Figure Lengend Snippet: ( A and B ) Flow nucleometry of RORγt ( A ) or phosphorylated Stat3 (Tyr705) ( B ) expression in nuclei isolated from in vitro–differentiated Th17 cells from WT and A20 ZF7 mice. Histograms are representative of n = 2–3 biologically independent experiments. ( C ) qPCR analyses of Il17a and Il22 expression in cells generated as in A . “Th17” indicates Th17 differentiation conditions. “FICZ” indicates treatment with Ahr agonist FICZ. ( D ) ELISA of IL-22 secretion from cells generated as in A . ( E ) ATAC-seq of genomic loci at or near the Il22 locus in cells generated as in A . ( F ) ChIP of acetylated H3K27 at indicated Il22 loci (loci a–c in E ) in cells generated as in A . ( G ) ChIP of histone H3 at the Il22 promoter and enhancer loci (loci a–c in E ). Cells were generated as in A and treated with the RORγt inhibitor GSK805. H3 ChIP directly assesses nucleosome occupancy and, thus, chromatin accessibility. ( H ) ChIP of acetylated H3K27 at Il22 enhancer loci (loci a–c in E ). Cells were generated as in A and treated with the RORγt inhibitor GSK805. ( I ) Flow cytometric analyses of RORγt expression in CRISPR/Cas9–edited primary human T cells differentiated in vitro using Th17 conditions. ( J ) qPCR analyses of expression of indicated genes in paired isogenic human Th17 cells engineered with CRISPR/Cas9 and either A20 ZF7 -targeted or nontargeting guide RNAs. Three pairs of isogenic samples from 2 healthy donors are shown. Data are shown as mean ± SEM. Statistics were calculated using unpaired 2-tailed Student’s t test with Welch’s correction ( C , D , and F ), 2-way ANOVA with post hoc Tukey’s multiple-comparison correction with simple effects ( G and H ), or paired-ratio t test ( J ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were subsequently differentiated into Th17 cells in plates precoated with 5 μg/mL anti-CD3 (Bio X Cell BE0001-2, clone OKT3) in Th17 medium (Immunocult XF serum-free medium with 50 μM β-mercaptoethanol, 30 ng/mL recombinant human IL-6 [Proteintech HZ-1019], 2.5 ng/mL recombinant human TGF-β1 [PeproTech 100-21], 10 ng/mL recombinant human IL-1β [Proteintech HZ-1164], 10 ng/mL recombinant human IL-23 [Proteintech HZ-1254], 10 μg/mL anti–IL-4 [Bio X Cell BE0240, clone MP4-25D2], and 10 μg/mL anti–IFN-γ [Bio X Cell BE0235, clone B133.5]) at a density of 1 million cells/mL.

Techniques: Expressing, Isolation, In Vitro, Generated, Enzyme-linked Immunosorbent Assay, CRISPR, Comparison