easyseptm mouse cd4+cd25+ treg cell isolation kit ii/easyseptm  (STEMCELL Technologies Inc)

Journal: PLOS ONE

Article Title: Obeticholic acid treatment ameliorates the cardiac dysfunction in NASH mice

doi: 10.1371/journal.pone.0276717

Figure Lengend Snippet: Correlation between ( A ) OCA-induced decrease in cardiac fibrosis (% of sirius red stained positive area) and ( B ) OCA-induced increase in fraction of shortening (FS) with OCA-induced increase in expressions of cardiac FXR/IL-10 levels, and infiltrated IL-10R+ Treg cells (CD4 + CD25 + ) in NASH mice heart. ( C ) Flow cytometry assessed intracellular FXR and NLRP3 levels in Ctrl and NASH group as well as percentile change in FXR and NLRP3 levels by OCA treatment. ( D ) Immunofluorescence-measured caspase-1 + cells (green color) in primary isolated splenic regulatory T (Treg cells) of Crtl and NASH group as well as percentile decrease in caspase-1 + and IL-1β + cells by OCA treatment. 20x, Scale bar: 100μm; **,***: p<0.01 or p<0.001 vs . Ctrl or NASH group.

Article Snippet: Mouse spleens from different groups were isolated using EasySepTM Mouse CD4 + CD25 + Treg cell isolation kit II/EasySepTM (StemCell Technologies, Vancouver, Canada).

Techniques: Staining, Flow Cytometry, Immunofluorescence, Isolation

Journal: PLOS ONE

Article Title: Obeticholic acid treatment ameliorates the cardiac dysfunction in NASH mice

doi: 10.1371/journal.pone.0276717

Figure Lengend Snippet: IF-based assessments of ( A ) Foxp3 + Ki-67 + (orange color), ( B ) Foxp3 + HLADR + (orange color), ( C ) TUNEL + (green color) ( D ) Annexin-5 + Pi + (green color) cells among primary CD4 + CD25 + Treg cells from Ctrl, Ctrl-OCA, or NASH, NASH-OCA mice. ( E ) Foxp3 + Ki-67 + , Foxp3 + HLADR + , TUNEL + , and Annexin-5 + Pi + cells among primary CD4 + CD25 + Treg cells from Ctrl and NASH mice. ( F ) percentile increase in Foxp3 + Ki-67 + , Foxp3 + HLADR + , and percentile decrease in TUNEL + , Annexin-5 + Pi + cells in primary CD4 + CD25 + Treg cells by OCA treatment. **,***: p<0.01 or p<0.001 vs . Ctrl or NASH group.

Article Snippet: Mouse spleens from different groups were isolated using EasySepTM Mouse CD4 + CD25 + Treg cell isolation kit II/EasySepTM (StemCell Technologies, Vancouver, Canada).

Techniques: TUNEL Assay

Journal: PLOS ONE

Article Title: Obeticholic acid treatment ameliorates the cardiac dysfunction in NASH mice

doi: 10.1371/journal.pone.0276717

Figure Lengend Snippet: Flow cytometry-assessed relative intracellular levels (/buffer group) of ( A ) FXR, IL-10R, IL-10, ( B ) NLRP3, caspase-1 among various CM pre-treated H9c2. Bar graph of ( C ) immunofluorescence-assessed Ki-67 + cells (red color), TUNEL + cells (green color), and ( D ) Annexin-5 + PI + (orange color) cells in all H9c2 cells. Scale bars in all images are 100 mm; CD4 + CD25 + Treg-NASH+OCA [supernatant of Treg cells from NASH mice as condition medium (cm) plus acute OCA incubation]; field of view (FOV).

Article Snippet: Mouse spleens from different groups were isolated using EasySepTM Mouse CD4 + CD25 + Treg cell isolation kit II/EasySepTM (StemCell Technologies, Vancouver, Canada).

Techniques: Flow Cytometry, Immunofluorescence, TUNEL Assay, Incubation

Journal: PLOS ONE

Article Title: Obeticholic acid treatment ameliorates the cardiac dysfunction in NASH mice

doi: 10.1371/journal.pone.0276717

Figure Lengend Snippet: ( A ) Expression of the differentiation markers [cardiac myosin heavy chain (MYH), myogenin, TNNI] levels (/Buffer group) in supernatant of H9c2 cells. ( B ) The collagen-1 and αSMA positive among H9c2 cells. ( C ) Collagen gel contraction assay for evaluating the function of cardiomyocyte after various pretreatment. Scale bar = 20 μm. CD4+CD25+ Treg-NASH+OCA [supernatant of Treg cells from NASH mice as condition medium (cm) plus acute OCA incubation].

Article Snippet: Mouse spleens from different groups were isolated using EasySepTM Mouse CD4 + CD25 + Treg cell isolation kit II/EasySepTM (StemCell Technologies, Vancouver, Canada).

Techniques: Expressing, Collagen Gel Contraction Assay, Incubation

Journal: Journal for Immunotherapy of Cancer

Article Title: PI3Kα inhibitor CYH33 triggers antitumor immunity in murine breast cancer by activating CD8 + T cells and promoting fatty acid metabolism

doi: 10.1136/jitc-2021-003093

Figure Lengend Snippet: CYH33 reshapes the immune profile in the TME. (A) CD45 + immune cells were enriched respectively from the single-cell suspension of four tumor tissues as presented in and subjected to scRNA-seq separately. UMAP plot of CD45 + immune cells from four tumor tissue samples was shown. (B) The percentage of each cluster in CD45 + immune cells was calculated. (C) The T cells from the enriched CD45 + cells in Balb/c mice were divided into six subclusters. The proportions of naïve CD8 + T cells (CD8 + Tn), effector CD8 + T cells (CD8 + Teff), memory CD8 + T cells (CD8 + Tm), exhausted CD8 + T cells (CD8 + Tex), helper CD4 + T cells (CD4 + Th), and regulatory CD4 + T cells (Treg) in CD3 + CD8 + T cells (left) or CD3 + CD4 + T cells (right) were presented. (D) The top-ranked enrichment pathways upregulated in CD3 + CD8 + T cells (left) or CD4 + Th cells (right) from enriched CD45 + cells in Balb/c mice using genes with a fold change greater than three after CYH33 treatment. (E) The percentage of each subcluster of CD11b + myeloid cells in CD45 + immune cells. cDCs, conventional dendritic cell; Krt18, keratin 18; MDSCs, myeloid-derived suppressor cells; pDC, plasmacytoid DCs; PMN, polymorphonuclear; scRNA-seq, single-cell RNA-seq; TME, tumor microenvironment; Treg, regulatory T cells; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: CD8 + T, CD4 + T, and CD4 + CD25 − T cells were enriched from cell suspension using EasySep Mouse CD8 + T cell Isolation Kit (Stemcell Technology, Vancouver, Canada, No. 19 853), Mouse CD4 + T cell Isolation Kit (Miltenyi Biotec, No. 130-104-453), or EasySep Mouse CD4 + CD25 + Tregs Isolation Kit II (Stemcell Technology, No. 18 783), respectively.

Techniques: Suspension, Derivative Assay, RNA Sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: PI3Kα inhibitor CYH33 triggers antitumor immunity in murine breast cancer by activating CD8 + T cells and promoting fatty acid metabolism

doi: 10.1136/jitc-2021-003093

Figure Lengend Snippet: CYH33 induces a proinflammatory TME and immunological memory. (A) Immunophenotyping of 4T1 (left, n=9 in vehicle group, n=8 in CYH33-treated group) and PY8119 (right, n=5) tumors by flow cytometry after CYH33 treatment for 7 days. The percentage of indicated immune cells in CD45 + cells was presented. (B) The frequency of Tregs (left) and the CD8/Treg ratio (right) in 4T1 tumors. (C) Representative images of 4T1 tumor tissue sections that were collected after CYH33 treatment for about 3 weeks and immunostained with the indicated markers. (D) The percentage of naïve (CD45RA + CD62L + CD44 − ), effector (CD45RA + CD62 − CD44 + ), effector memory (CD45RA − CD62 − CD44 + , TEM), and central memory (CD45RA − CD62L + CD44 + , TCM) in the tumor-infiltrating CD8 + T cells (left, n=9) or CD4 + T cells (right, n=9). (E) The percentage of CD8 + T cells (above) or CD4 + T cells (bottom) positive of IFN-γ, TNF-α, or granzyme B (n=5). (F) The schedule of the tumor rechallenging experiment (above). The tumor volume was measured (bottom). Data are presented as mean±SEM. IFN-γ, interferon-γ; TCM, central memory T cell; TME, tumor microenvironment; TNF-α, tumor necrosis factor-α; Tregs, regulatory T cells. *: p < 0.05, **: p < 0.01, ****: p < 0.0001.

Article Snippet: CD8 + T, CD4 + T, and CD4 + CD25 − T cells were enriched from cell suspension using EasySep Mouse CD8 + T cell Isolation Kit (Stemcell Technology, Vancouver, Canada, No. 19 853), Mouse CD4 + T cell Isolation Kit (Miltenyi Biotec, No. 130-104-453), or EasySep Mouse CD4 + CD25 + Tregs Isolation Kit II (Stemcell Technology, No. 18 783), respectively.

Techniques: Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: PI3Kα inhibitor CYH33 triggers antitumor immunity in murine breast cancer by activating CD8 + T cells and promoting fatty acid metabolism

doi: 10.1136/jitc-2021-003093

Figure Lengend Snippet: CYH33 inhibits 4T1 tumor progression via CD8 + T cells and abrogates M2 macrophage-mediated suppression. (A) CD8 + T cells, CD4 + T cells, or macrophages were depleted in 4T1-bearing Balb/c mice by injection of anti-CD8 Abs, anti-CD4 Abs or clodrosome, respectively. The mice were administered with CYH33 for the indicated time. Data are presented as mean±SEM. (B) The CD8 + T cells and CD4 + T cells were isolated from spleens of naïve Balb/c mice and stimulated with anti-CD3 and anti-CD28. The Tregs were induced from CD4 + CD25 - T cells and stimulated with dynabeads coated with anti-CD3 and anti-CD28. The proliferation of CD8 + T cells (left), CD4 + T cells (middle) and Tregs (right) in the presence of CYH33 was determined by measuring the content of carboxyfluorescein succinimidyl amino ester (CFSE) with flow cytometry. Representative histograms depicting dividing cells were presented. The percentage of the dividing populations was indicated. The results were from two independent experiments. (C) The M0, M1 or M2-polarized BMDMs were induced by CSF-1, CSF-1 +LPS or CSF-1 +IL-4, respectively, for 24 hours and then treated with CYH33 for 72 hours. Cell viability was evaluated by the CCK-8 assay. (D) BMDMs were primed with LPS or IL-4 to induce polarization towards M1 (left) or M2 (right), respectively, in the presence of CYH33 at 1 µM. Total RNA were extracted 24 hours later and qPCR was performed to detect the mRNA level of indicated genes. (E) CD8 + T cells labeled with CFSE were cocultured with M2-primed macrophages for 72 hours in the presence of CYH33. Representative histograms were shown (left) and percentage of dividing cells was calculated (right). (F) The mRNA level of indicated genes in macrophages cocultured with CD8 + T cells was determined by qPCR. Data are presented as mean±SD from two independent experiments. IL-4, interleukin 4; qPCR, quantitative PCR; RTV, relative tumor volume; Treg, regulatory T cells. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Article Snippet: CD8 + T, CD4 + T, and CD4 + CD25 − T cells were enriched from cell suspension using EasySep Mouse CD8 + T cell Isolation Kit (Stemcell Technology, Vancouver, Canada, No. 19 853), Mouse CD4 + T cell Isolation Kit (Miltenyi Biotec, No. 130-104-453), or EasySep Mouse CD4 + CD25 + Tregs Isolation Kit II (Stemcell Technology, No. 18 783), respectively.

Techniques: Injection, Isolation, Flow Cytometry, CCK-8 Assay, Labeling, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: PI3Kα inhibitor CYH33 triggers antitumor immunity in murine breast cancer by activating CD8 + T cells and promoting fatty acid metabolism

doi: 10.1136/jitc-2021-003093

Figure Lengend Snippet: CYH33 treatment augments FA metabolism to boost antitumor immunity and synergizes with a FASN inhibitor. (A) The Balb/c mice bearing 4T1 tumors were treated with vehicle or CYH33 for 4 days and tumors were subjected to RNA-seq. Gene set enrichment analysis (GSEA) enrichment plots of differentially expressed genes in the gene set of FA metabolism and adipogenesis were presented. Red or blue represents positive and negative enrichment, respectively. (B) The transcripts per million of indicated genes involving in FA metabolism were presented (n=4). (C) The mRNA level of indicated genes from 4T1 allografts after CYH33 treatment for 4 days was measured by qPCR (n=5). (D) The mRNA level of indicated genes from 4T1 cells treated with 1 µM CYH33 for 24 hours was measured by qPCR. The results were from three independent experiments. (E) The level of FFA in the culture medium of 4T1 cells after treatment with 1 µM CYH33 for 72 hours from three independent experiments. (F) The expression of IFN-γ, TNF-α, and granzyme B in CD8 + T cells cultured in the glucose-free 1640 medium with or without FFA supplementation were determined by flow cytometry. The results were presented as the fold change of median fluorescence intensity from two independent experiments. (G) Balb/c mice bearing 4T1 allografts were administered with vehicle control, CYH33 (20 mg/kg, once a day), C75 (10 mg/kg, once every 3 days), or combination of CYH33 and C75 for 17 days. The relative tumor volumes are presented as mean±SEM. (H) Tumor tissues were collected at the end of experiment and the level of FFA was measured from the lipid extraction (n=7). (I) Representative images of 4T1 tumor sections immunostained with Ki67, cleaved-caspase 3, CD4, CD8, F4/80 and CD206. FA, fatty acid; FFA, free fatty acid; FASN, FA synthase; IFN-γ, interferon-γ; qPCR, quantitative PCR; RTV, relative tumor volume; TNF-α, tumor necrosis factor-α. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: CD8 + T, CD4 + T, and CD4 + CD25 − T cells were enriched from cell suspension using EasySep Mouse CD8 + T cell Isolation Kit (Stemcell Technology, Vancouver, Canada, No. 19 853), Mouse CD4 + T cell Isolation Kit (Miltenyi Biotec, No. 130-104-453), or EasySep Mouse CD4 + CD25 + Tregs Isolation Kit II (Stemcell Technology, No. 18 783), respectively.

Techniques: RNA Sequencing, Expressing, Cell Culture, Flow Cytometry, Fluorescence, Control, Extraction, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: RGC-32 Promotes Th17 Cell Differentiation and Enhances Experimental Autoimmune Encephalomyelitis

doi: 10.4049/jimmunol.1602158

Figure Lengend Snippet: RGC-32 is preferentially induced in Th17 cells and promotes their differentiation. (A) Quantitative RT-PCR for RGC-32 in naive CD4+ T cells from spleens of WT mice stimulated for 48 h with anti-CD3 and anti-CD28 in the presence or absence of indicated cytokines and in (B) Th0, Th1, Th2, Th17, or Treg polarizing conditions (**p, 0.01; mean 6 SEM; n = 3–4 mice per group). (C) Representative histogram of RGC-32 intracellular staining in CD4+ T cells cultured under Th17 and Treg conditions. Gray histogram represents background staining in RGC-32−/− CD4+ T cells. (D and E) Naive CD4+ T cells were cultured for 72 h in Th17 conditions and intracellular expression of IL-17A was assessed by flow cytometry. A representative profile is shown. Plots were gated on viable singlet CD4+ events. (F) ELISA for IL-17A determined in supernatants. (G) Real-time PCR for IL-17A. (H) Quantitative RT-PCR for Th17 signature genes IL-17F, IL-21, IL-22, and IL-23R. (*p, 0.05, **p, 0.01, mean 6 SEM). Data are representative of more than three independent experiments (n = 3–4 mice per group).

Article Snippet: CD4 + CD25 + Tregs were purified using a mouse Treg isolation kit II (Stemcell Technologies).

Techniques: Quantitative RT-PCR, Staining, Cell Culture, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: RGC-32 Promotes Th17 Cell Differentiation and Enhances Experimental Autoimmune Encephalomyelitis

doi: 10.4049/jimmunol.1602158

Figure Lengend Snippet: RGC-32−/− CD4+ T cells polarize normally to Th1, Th2, and Treg lineages. Naive CD4+ T cells were skewed in vitro under Th1, Th2, and Treg conditions. (A) Intracellular expression of IFN-γ was assessed by flow cytometry. (B) ELISA for IFN-γ determined in supernatants. (C) Intracellular expression of IL-4 was assessed by flow cytometry. (D) ELISA for IL-4 determined in supernatants. (E) Foxp3 expression was determined by intracellular staining. (F) In vitro suppression assay of purified CD4+CD252 T responder cells cocultured for 3 d with in vitro–differentiated, purified CD4+CD25+ iTregs from WT or RGC-32−/− mice. Proliferation was assessed by [3H]thymidine incorporation added in the last 18 h of culture (left panel). Percentage inhibition at 1:1 and 1:2 Treg/T responder ratio is shown in the right panel. ***p, 0.001. Data represent the mean 6 SD and are representative of more than three independent experiments (n = 3 mice per group). Representative profiles shown for intracellular IL-4, IFN-γ, and Foxp3 are gated on viable singlet CD4+ events.

Article Snippet: CD4 + CD25 + Tregs were purified using a mouse Treg isolation kit II (Stemcell Technologies).

Techniques: In Vitro, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Suppression Assay, Purification, Inhibition