3154019b  (fluidigm)

Journal: Cell reports

Article Title: Neutral ceramidase-dependent regulation of macrophage metabolism directs intestinal immune homeostasis and controls enteric infection

doi: 10.1016/j.celrep.2022.110560

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD73 (154 Sm) , Fluidigm , Cat# 3154019B; RRID: AB_2813854.

Techniques: Recombinant, Western Blot, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Cell Culture, IP Phosphatase Assay, Software

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: A negative feedback loop between fibroadipogenic progenitors and muscle fibres involving endothelin promotes human muscle fibrosis

doi: 10.1002/jcsm.12974

Figure Lengend Snippet: Antibody panel used for CyTOF

Article Snippet: CD73 , 168Er , AD2 , Fluidigm , 3168015B.

Techniques:

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: A negative feedback loop between fibroadipogenic progenitors and muscle fibres involving endothelin promotes human muscle fibrosis

doi: 10.1002/jcsm.12974

Figure Lengend Snippet: Human FAPs isolated from fibrotic muscles differ from those isolated from nonfibrotic muscles. ( A ) Uniform manifold approximation and projection (UMAP) map showing the distributions of cells from M CT (yellow), FibM CT (orange), and FibM OP (red) muscle biopsies. For each condition, nonmyogenic cells were extracted, and data from three to four patients were concatenated. Each dot represents a single cell, and 300 000 cells were used to obtain the map. The 37 markers used for the analysis are listed in Table . ( B ) UMAP plots showing the expression patterns of PDGFRα, CD90, CD105, and CD73 in nonmyogenic cells from M CT , FibM CT , and FibM OP muscle biopsies. Cells are coloured according to the intensity of the marker shown. ( C ) Expression dot plot of selected markers from the CyTOF analysis of nonmyogenic cells from fibrotic and nonfibrotic muscles. Dots are coloured according to the average intensity with which the marker was expressed, and the size of each dot shows the percentage of nonmyogenic cells expressing each marker in each condition: M CT , FibM CT , and FibM OP . ( D ) Adipogenic and osteogenic differentiation of human FAPs isolated from fibrotic or nonfibrotic muscles. Left panel: Oil red O staining of FAPs from M CT , FibM CT , and FibM OP muscle biopsies in adipogenic differentiation medium. Right panel: Alizarin red staining of FAP cells from M CT , FibM CT , and FibM OP muscle biopsies in osteogenic differentiation medium.

Article Snippet: CD73 , 168Er , AD2 , Fluidigm , 3168015B.

Techniques: Isolation, Muscles, Expressing, Marker, Staining

Journal: Cell stem cell

Article Title: Smooth muscle cell reprogramming in aortic aneurysms

doi: 10.1016/j.stem.2020.02.013

Figure Lengend Snippet: (A) Representative IMC images stained for GFP (green), CD105 (red), CD73 (blue), CD90 (yellow), Sca-1 (magenta), and CD44 (cyan) in the overlaid format and individual channel images (N=6 mice/group). Scale bar: 500 μm. (B) PhenoGraph clustering of all cell phenotype visualized as a distinct color on the tSNE plot. (C) tSNE plot of high-dimension single cell data. (D) Heatmap visualizing the marker intensity for each Phenograph. (E) (left) Cluster 17 location in the tissue section using HistoCAT spatial clustering analysis. (right) A high-magnification of (Ea) left. (F) A cartoon showing confetti color distribution in bone nodule area and adjacent to the bone nodule area. (G) TGFβR2iSMC-Apoe mice VSMCs adjacent to the bone nodule area are mixed labeled with red fluorescent protein (RFP), yellow fluorescent protein (YFP), nuclear (n) green fluorescent protein (GFP), or membrane associated (m) cyan fluorescent protein (CFP). Scale bar: 10 μm. (H) TGFβR2iSMC-Apoe mice VSMCs in the bone nodule media areas are labeled with single color fluorescent protein after 4 months of high cholesterol high fat diet. Scale bar: 10 μm. (I) Bar chart showing the proportions of each of the Confetti colors in TGFβR2iSMC-Apoe mice VSMCs in media ascending aorta adjacent to the bone nodule area and in bone nodule area. N=10 for TGFβR2iSMC-Apoe mice. See also Figure S4, Figure S5, and Table S2B.

Article Snippet: Rat monoclonal anti-CD73-154Sm (clone TY/11.8) , Fluidigm , Cat# 3154019B, RRID:AB_2813854.

Techniques: Staining, Marker, Labeling

Journal: Cell stem cell

Article Title: Smooth muscle cell reprogramming in aortic aneurysms

doi: 10.1016/j.stem.2020.02.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat monoclonal anti-CD73-154Sm (clone TY/11.8) , Fluidigm , Cat# 3154019B, RRID:AB_2813854.

Techniques: Recombinant, Multiplex Assay, Staining, In Situ, Marker, Expressing, Plasmid Preparation, shRNA, Software

Journal: Cell reports

Article Title: T-bet+ Memory B Cells Link to Local Cross-Reactive IgG upon Human Rhinovirus Infection

doi: 10.1016/j.celrep.2019.12.027

Figure Lengend Snippet:

Article Snippet: Anti-human CD73-168Er (CyTOF) , Fluidigm , Cat#3168015B; RRID:AB_2810249.

Techniques: In Vitro, Blocking Assay, Multiplex Assay, Virus, Infection, Recombinant, Next-Generation Sequencing, RNA Sequencing, Software, Microscopy, Imaging, Conjugation Assay, Staining

Journal: Circulation research

Article Title: Atherosclerosis-Driven Treg Plasticity Results in Formation of a Dysfunctional Subset of Plastic IFNγ + Th1/Tregs

doi: 10.1161/CIRCRESAHA.116.309764

Figure Lengend Snippet: (A) Experimental design. 27 week-old Apoe−/− littermates received 1×106 CTV-labeled Mir146a−/− or Mir146a+/+ Tregs and were subsequently placed on a WD regimen for 8 weeks (n= 5 male- and 3-4 female- Apoe−/− recipients/cohort). On week 4, both cohorts received an additional boost of 1×106 FarRed+ Mir146a+/+ or Mir146a−/− Tregs. Eight weeks later, the aortas, carotid arteries, lymphoid organs, and blood were collected. (B) Percentage of donor and IFNγ+ Mir146a+/+ and Mir146a−/− Tregs within 2-3 concatenated recipient Apoe−/− carotids (n=3 concatenated samples/genotype). (C) Representative Mir146a+/+ (Green squares) and Mir146a−/− (Red squares) Treg recipient Apoe−/− Oil Red O staining and lesion percentage of the aorta. (D) Representative MOVAT staining and the percentage of aortic root lesions in Mir146a+/+ and Mir146a−/− Treg recipient mice. (E-I) Representative flow cytometry results for the Treg recipient Apoe−/− carotid arteries. (E) The percentage and number of CD45+ leukocytes and CD45+CD4+ T cells within the Apoe−/− recipient carotid artery (two carotid arteries/recipient). (F, G) Representative host CD4+ T cell IFNγ FACS staining and quantification within the Apoe−/− Treg recipient carotids. (H, I) Carotid artery F4/80+MHC-II+ macrophage representative flow cytometry staining and quantification within the Apoe−/− recipients (n= 7-8 Apoe−/− recipients/Treg population, three independent experiments. (J, K) Mir146a−/− Tregs readily undergo plasticity in response to IL-27, IL-12, and IFNγ. CD25+ Mir146a+/+ and Mir146a−/− Tregs were isolated and cultured in complete RPMI1640 with CD3/CD28 antibodies, and IL-2 (50ng/ml, Vehicle), or IL-2, IL-12 (25ng/ml), IL-27 (25ng/ml), and IFNγ (25ng/ml) for 4 days and assessed for IFNγ+Foxp3+ Tregs Representative flow cytometry plots, (J) fold induction of IFNγ+ Tregs vs the Mir146a+/+ Treg vehicle controls. n = 3 independent experiments. The mean±SEM is shown. *-p<0.05, **-p<0.01, NS-Not significant, unpaired, one-tailed Bonferroni-Holm-corrected student’s T tests.

Article Snippet: While the elevated Ebi3 expression data are interesting and may potentially reflect a compensatory mechanism, our flow cytometry, RNAseq , RT-PCR phenotyping and cellular assays of Th1/Tregs is consistent with a non-suppressive role for these cells, suggesting that the upregulation of Ebi3 is insufficient to restore the suppressive functionality to Th1/Tregs. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 7 caption a7 caption a8 scRNA-seq reveals down-regulation of multiple Treg immunosuppressive genes within plastic Th1/Tregs Sorted 40 week old Apoe −/− splenic CD4 + CD73 + PD1 + CD25 + CCR5 + T cells were used to create single cell libraries for Fluidigm C1-based SMARTer RNA-Seq ( Supplemental Fig. 3 , n=3-4 mice/experiment, 6 experiments, 270 single T cell libraries).

Techniques: Labeling, Staining, Flow Cytometry, Isolation, Cell Culture, One-tailed Test

Journal: Circulation research

Article Title: Atherosclerosis-Driven Treg Plasticity Results in Formation of a Dysfunctional Subset of Plastic IFNγ + Th1/Tregs

doi: 10.1161/CIRCRESAHA.116.309764

Figure Lengend Snippet: Forty week old Apoe−/− spleens (shown), PALNs, and aortas (not shown) were isolated and re-stimulated for ICS. CD45+CD4+Foxp3+ Tregs were gated and IFNγ+Foxp3+ (red histograms) and IFNγ− Foxp3+ (blue histograms) subgates were examined for the expression of the Treg markers GITR (A), CD25 (B), CD73 (C), and PD1 (D), and the Th1 markers Tbet (E), CXCR3 (F), CD119 (G), and CCR5 (H). All gates were set using isotype controls (gray histogram) and the percentages of positive cells are shown (means±SEM). (I-J) Aged Apoe−/− and C57Bl6 spleens (shown), PALNs, and aortas (not shown) were re-stimulated for ICS. CD45+CD4+Foxp3+ Tregs were gated and examined for IFNγ and CXCR3 expression. (J) The percentages of IFNγ and CXCR3 subsets amongst Foxp3+ Tregs from aged Apoe−/− and C57Bl6 spleens. The means±SEM are shown. n=8 mice, 4 independent experiments. ***-p<0.001, **-p<0.01, *-p<0.05, unpaired student’s T test.

Article Snippet: While the elevated Ebi3 expression data are interesting and may potentially reflect a compensatory mechanism, our flow cytometry, RNAseq , RT-PCR phenotyping and cellular assays of Th1/Tregs is consistent with a non-suppressive role for these cells, suggesting that the upregulation of Ebi3 is insufficient to restore the suppressive functionality to Th1/Tregs. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 7 caption a7 caption a8 scRNA-seq reveals down-regulation of multiple Treg immunosuppressive genes within plastic Th1/Tregs Sorted 40 week old Apoe −/− splenic CD4 + CD73 + PD1 + CD25 + CCR5 + T cells were used to create single cell libraries for Fluidigm C1-based SMARTer RNA-Seq ( Supplemental Fig. 3 , n=3-4 mice/experiment, 6 experiments, 270 single T cell libraries).

Techniques: Isolation, Expressing

Journal: Circulation research

Article Title: Atherosclerosis-Driven Treg Plasticity Results in Formation of a Dysfunctional Subset of Plastic IFNγ + Th1/Tregs

doi: 10.1161/CIRCRESAHA.116.309764

Figure Lengend Snippet: Splenic CD4+ T cells were isolated from aged Apoe−/−, Foxp3eGFP, or Foxp3Yfp-cre/Yfp-creR26RtdTomato/tdTomato Apoe−/− and Foxp3Yfp-cre/Yfp-creR26RtdTomato/tdTomato C57Bl6 mice and the following subsets were purified for T cell suppression assays: CCR5− Tregs, CCR5+ Th1/Tregs, Foxp3eGFP+ Tregs, CCR5+ Th1, CD4+Foxp3− CFSE-labeled T responders (Tresp), and CD4-depleted splenocytes (APCs). (A-K) Representative IFNγ, Foxp3, CCR5, or isotype control staining. Isotype controls for IFNγ (A), and CCR5 (G). IFNγ staining within CD4+Foxp3+-gated Apoe−/− CCR5+ Th1/Treg (B), CCR5− Treg (C), C57Bl6 Foxp3GFP+ Treg (D), Apoe−/− CCR5+ Th1 (E) post sort isolates, and the CD4+ Apoe−/− pre-sort population (F). (H-K) Representative CCR5 staining for CCR5+ Th1/Treg (H), CCR5− Treg (I), C57Bl6 Foxp3GFP+ Treg (J), and CCR5+ Th1 (K) post sort isolates. (L,M,N) T cell suppression assay results. Comparison of (L) Apoe−/− and C57Bl6 CCR5− Tregs, (M) C57Bl6 CCR5− Tregs, C57Bl6 CCR5− Tregs spiked with Apoe−/− Th1 cells, and Apoe−/− CCR5+ Th1/Tregs in the suppression assays. (N) The mean suppressive ability of Apoe−/− CCR5− Tregs and Apoe−/− CCR5+ Th1/Tregs as a percentage of C57Bl6 CCR5− Treg suppression (n=6 independent experiments). (O) Sorted CD4+Foxp3+CCR5+ Tregs (Treg/Th1) and CD4+Foxp3+CCR5− Treg cells from aged 40 week-old chow diet Foxp3Yfp-cre/Yfp-CreR26RtdTomato/tdTomatoApoe−/− mice were processed for Il10, Tgfβ, and Ebi3 expression (n=4/ per cell type). The mean±SEM is shown. **-p<0.01, NS–not significant, Bonferroni-holm-corrected unpaired student’s T tests.

Article Snippet: While the elevated Ebi3 expression data are interesting and may potentially reflect a compensatory mechanism, our flow cytometry, RNAseq , RT-PCR phenotyping and cellular assays of Th1/Tregs is consistent with a non-suppressive role for these cells, suggesting that the upregulation of Ebi3 is insufficient to restore the suppressive functionality to Th1/Tregs. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 7 caption a7 caption a8 scRNA-seq reveals down-regulation of multiple Treg immunosuppressive genes within plastic Th1/Tregs Sorted 40 week old Apoe −/− splenic CD4 + CD73 + PD1 + CD25 + CCR5 + T cells were used to create single cell libraries for Fluidigm C1-based SMARTer RNA-Seq ( Supplemental Fig. 3 , n=3-4 mice/experiment, 6 experiments, 270 single T cell libraries).

Techniques: Isolation, Purification, Labeling, Control, Staining, Suppression Assay, Comparison, Expressing

Journal: Circulation research

Article Title: Atherosclerosis-Driven Treg Plasticity Results in Formation of a Dysfunctional Subset of Plastic IFNγ + Th1/Tregs

doi: 10.1161/CIRCRESAHA.116.309764

Figure Lengend Snippet: Sorted 40 week old Apoe−/− splenic CD4+CD73+PD1+CD25+CCR5+ T cells were used to create single cell libraries for Fluidigm C1-based SMARTer RNA-Seq (Supplemental Fig. 3, n=3-4 mice/experiment, 6 experiments, 270 single T cell libraries). The sequenced, processed single T cell libraries were hierarchically clustered into Th1 (red, n=83), Treg (green, n=21), and Th1/Treg (blue, n=72) cells and analyzed for differential gene expression. (A) Select significantly different violin plots are shown for each subset. (B-C) Summary of the IPA-based re-sorting and clustering. (B) A Venn diagram of genes with an expression level above 100 reads, and IPA generated connectivity among the categories of unique and shared genes for clades 1-3. (C) The 537 genes uniquely expressed by clade 1 were used to generate a predicted network that is highly expressed in Th1/Tregs. The average expression levels among a representative set of clade 1 cells is overlaid, with the intensity of red showing denoting higher FPKM counts. ***-p<0.001, **-p<0.01, *-p<0.05, NS–not significant, Tukey ANOVA post-hoc tests.

Article Snippet: While the elevated Ebi3 expression data are interesting and may potentially reflect a compensatory mechanism, our flow cytometry, RNAseq , RT-PCR phenotyping and cellular assays of Th1/Tregs is consistent with a non-suppressive role for these cells, suggesting that the upregulation of Ebi3 is insufficient to restore the suppressive functionality to Th1/Tregs. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 7 caption a7 caption a8 scRNA-seq reveals down-regulation of multiple Treg immunosuppressive genes within plastic Th1/Tregs Sorted 40 week old Apoe −/− splenic CD4 + CD73 + PD1 + CD25 + CCR5 + T cells were used to create single cell libraries for Fluidigm C1-based SMARTer RNA-Seq ( Supplemental Fig. 3 , n=3-4 mice/experiment, 6 experiments, 270 single T cell libraries).

Techniques: RNA Sequencing, Gene Expression, Expressing, Generated