uea1 dylight647 conjugated lectin  (Vector Laboratories)

Journal: The Journal of Experimental Medicine

Article Title: Neonatal microbiota colonization primes maturation of goblet cell–mediated protection in the pre-weaning colon

doi: 10.1084/jem.20241591

Figure Lengend Snippet: senGC maturation is microbiota dependent. (A) Ex vivo mucus growth in adult GF and ConvR mouse colon after stimulation with bacterial MAMPs. (B) Ex vivo mucus growth dose response to P3CSK4 in adult GF and ConvR mouse colon. (C) Ex vivo mucus growth in adult GF and ConvR mouse colon stimulated with P3CSK4 in the presence or absence of senGC activation inhibitors. (D) AB/PAS-stained tissue sections from ex vivo experiments illustrated in A. Emptied upper crypt GCs (red arrowheads) and lower crypt cavitation (yellow arrowheads) indicated. (E) Whole-mount confocal imaging of adult GF mouse colon treated with fluorescent dextran tracer. Images show x/y-axis (upper panel) and x/z-axis (lower panel) cross-sections illustrating dextran uptake by an upper crypt GC (purple arrowhead). (F) Ex vivo mucus growth in neonatal (P3, 5, and 15) and postweaning (P33) rat colon stimulated with P3CSK4 in the presence or absence of Dynasore inhibitor. (G) Standardized expression of genes (columns) encoding known and predicted secreted proteins upregulated in mucus from P9-adult compared with P1–P7 rats (see ) in GC subpopulations (rows) identified by scRNA-seq. “Secretion” row indicates evidence of secretion determined by prior annotation or in silico predication of classical or nonclassical secretion by SecretomeP. (H) Quantification of the frequency of Tgm3-expressing GCs as a proportion of the total GC population in neonatal (P3, P9, P14, and P19) and postweaning (P24) colonic tissue sections from ConvR mice. (I) Confocal micrographs of representative tissue sections from P3 and P14 ConvR mice stained for Tgm3 (green) or the epithelial border and GC-binding lectin WGA (grey) and the GC-specific lectin UEA1 (red). The epithelial surface (blue dashed line) and an individual GC from each image is indicated (yellow dashed line). Data represent n = 3–5 (A–F, H, and I) animals per group, as indicated. All data are pooled from at least two independent litters or experiments. All error-bar graphs show median and interquartile range. Statistical comparisons between groups by two-way ANOVA and Fisher’s LSD (A and C) or Kruskall–Wallis and uncorrected Dunn’s test (F and H); P < 0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****). Image scale bars are 50 µm (D and I) or 20 µm (E). # note: ConvR data displayed in A and B are reproduced from our previous publication and are shown for illustrative purposes only.

Article Snippet: Lastly, slides were washed with PBS and counterstained with a Hoechst-34580 DNA dye (5 μg/ml; Merck) in some cases supplemented with combinations of Ulex europaeus agglutinin I (UEA1) Atto 488–conjugated lectin (10 µg/ml; Merck), UEA1 DyLight647–conjugated lectin (10 µg/ml; Vectorlabs), or Wheat germ agglutinin (WGA) Alexa 555–conjugated lectin (10 µg/ml; Thermo Fisher Scientific) for 15 min.

Techniques: Ex Vivo, Activation Assay, Staining, Imaging, Expressing, In Silico, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Neonatal microbiota colonization primes maturation of goblet cell–mediated protection in the pre-weaning colon

doi: 10.1084/jem.20241591

Figure Lengend Snippet: Microbiota induction of senGC maturation via regulation of Duox2. (A) Schematic of the senGC activation pathway highlighting known (black) and putative (red) pathway genes. (B) Expression of known and putative senGC genes in FACS-isolated colonic GCs and colonocytes determined by DESeq2 analysis of bulk RNA sequencing data. (C) Comparison of gene expression ratios between P22 ConvR:GF mice and adult 3-wk ConvD:GF mice quantified by DESeq2 analysis of bulk colonic RNA sequencing data. Genes significantly upregulated (red) or downregulated (blue) by microbiota exposure in both P22 and ConvD mice are indicated. (D) Proportion of unique and shared genes significantly regulated by microbiota exposure in P22 ConvR and adult 3-wk ConvD mice, based on data shown in C. (E) Comparison of microbiota-dependent expression of known and putative senGC activation pathway genes (A and B) in P22 ConvR and adult 3-wk ConvD mice. Subset of data shown in C. Genes not significantly regulated by microbiota in either group (grey) or genes regulated in either P22 ConvR (purple), adult ConvD (yellow), or both groups (teal) are indicated. (F) Relative expression (compared with GF) of Duox2 (left) and Nox1 (right) genes in ConvD (brown) and B. fragilis monoassociated (blue) mice from 1 to 4 wk (w) colonization. Expression determined by qRT-PCR of colonic RNA, normalized to Gapdh and Rplp0 expression. (G) Expression of Duox2 (left) and Nox1 (right) genes in postnatal ConvR (purple; P3–33) and GF (teal; P9–P33) determined by DESeq2 analysis of bulk colonic RNA sequencing data. (H) Confocal micrographs of fixed colonic tissue sections from ConvR WT mice stained for Duox2 (left) and Nox1 (right) mRNA by in situ RNA hybridization and counterstained by Epcam (grey). Duox2- or Nox1-expressing crypt regions are indicated (yellow arrowheads). (I) Confocal micrographs showing upper crypt GCs in fixed colonic tissue sections from ConvR, GF, and ConvD mice stained for Duox2 (red), mucus (UEA1; green), actin (grey), and DNA (blue). Intracellular Duox2 in GCs are indicated (yellow arrowheads). (J) Ex vivo mucus growth in Duox2 fl/fl and Duox2 ΔIEC colon tissue treated with carbachol (CCh), LPS, or P3CSK4. (K) Ex vivo mucus growth in WT colon tissue treated with P3CSK4 in the presence or absence of the Nox1 inhibitor ML171. Data represent n = 2–5 animals per group, as indicated. All data are pooled from at least two independent experiments or litters. All error-bar graphs show median and interquartile range. Statistical comparisons between groups by DESeq2 (B, C, and E), Kruskal–Wallis and Dunn’s multiple comparison (F), or two-way ANOVA and Fisher’s LSD (J and K); P < 0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****). Scale bars are 50 µm (H) or 5 µm (I). FC, fold change.

Article Snippet: Lastly, slides were washed with PBS and counterstained with a Hoechst-34580 DNA dye (5 μg/ml; Merck) in some cases supplemented with combinations of Ulex europaeus agglutinin I (UEA1) Atto 488–conjugated lectin (10 µg/ml; Merck), UEA1 DyLight647–conjugated lectin (10 µg/ml; Vectorlabs), or Wheat germ agglutinin (WGA) Alexa 555–conjugated lectin (10 µg/ml; Thermo Fisher Scientific) for 15 min.

Techniques: Activation Assay, Expressing, Isolation, RNA Sequencing, Comparison, Gene Expression, Quantitative RT-PCR, Staining, In Situ, Hybridization, Ex Vivo

Journal: Nature Immunology

Article Title: Age-related epithelial defects limit thymic function and regeneration

doi: 10.1038/s41590-024-01915-9

Figure Lengend Snippet: a , Uniform Manifold Approximation and Projection (UMAP) of 22,932 CD45 − thymic cells from 2-mo and 18-mo female C57BL/6 mice, annotated by cell type subset and outlined by cell compartment (epithelial; fibroblast; endothelial; MEC; vSMC/PC; nmSC). b , ThymoSight integration of public data for murine nonhematopoietic thymic stromal cells, including our own dataset ( n = 297,988) annotated by publication source and outlined by cell type and compartment. c , Violin plots highlighting key genes marking individual subsets within individual structural compartments (fibroblast, endothelium and epithelium). d , e , UMAPs of individual structural compartments color-coded by cell type subset ( d ) and age cohort ( e ). n EC = 1,661; n FB = 13,240; n TEC = 6,175. f , Scaled change in frequency for each individual structural cell subset with age. g , Gating strategy and quantities for cell populations within the epithelial lineage (based on previous work ) in 2-mo ( n = 10) and 18-mo ( n = 10) mice. First, based on a CD45 − EpCAM + parent gate, tuft cells were identified by expression of L1CAM, then all other TECs were assessed for expression of conventional TEC markers UEA1 and Ly51. Within the UEA1 hi Ly51 lo mTEC population CD104 + MHCII lo cells were identified as mTEC1. Cells that were deemed as non-mTEC1 were then fractionated based on MHCII and Ly6D. h , Concatenated flow cytometry plots and graphs highlighting the frequency of Ly51 − UEA1 − (DN-TECs) across lifespan (gated on CD45 − EpCAM + MHCII + cells). i , Violin plots of aaTEC1 and aaTEC2 novel markers. j , k , Flow cytometry plots ( j ) and quantities ( k ) for aaTEC1 and aaTEC2 populations in 2-mo ( n = 15), 12-mo ( n = 10) or 18-mo ( n = 13) male and female C57BL/6 mice. Summary data represent mean ± s.e.m.; each dot represents an individual biological replicate. Statistics were generated using a two-tailed Mann–Whitney test comparing within individual subsets ( g ) or Kruskal–Wallis ( k ) test with Dunn’s correction.

Article Snippet: The antibodies used were rabbit anti-pan-cytokeratin (Dako, cat. no. Z0622), anti-K5 (BioLegend, cat. no. poly19055), rat anti-mouse K8/18 (Troma-1; Developmental Studies Hybridoma Bank), rabbit anti-K14 (Abcam, cat. no. EPR17350), rat anti-mouse AIRE (WEHI, clone 5H12), rabbit anti-human/mouse DCLK1 (LSBio, cat. no. LS-C100746) and biotinylated UEA1 lectin (Vector Labs, cat. no. B-1065).

Techniques: Expressing, Flow Cytometry, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Nature Immunology

Article Title: Age-related epithelial defects limit thymic function and regeneration

doi: 10.1038/s41590-024-01915-9

Figure Lengend Snippet: a–c , Concatenated flow cytometry plots and quantities for cell populations within the fibroblast ( a ), endothelial ( b ), and “other” ( c ) cell lineages in 2-mo (n = 10) and 18-mo (n = 10) mice. c , Concatenated flow cytometry plots and quantities for pericytes (PC), vascular smooth muscle cells (vSMC) and mesothelial cells (MEC) (n = 10/age). d , Frequency and numbers of DN-TEC across lifespan: 2-mo (n = 14), 6-mo (n = 5), 9mo (n = 15), 12-mo (n = 5), and 18+mo (n = 18). e , Violin plots with extensive list of aaTEC1 and aaTEC2 markers. f , Gating strategy for aaTECs. aaTEC1 were first gated on CD45 − TER119 − then PDGFRα - CD31 − cells. EpCAM + MHCII + cells were gated as the whole TEC compartment, then mTECs and cTECs were excluded by taking the UEA1 − Ly51 − double negative fraction and gating on CLDN3. aaTEC2 were also first gated on CD45 − TER119 − then PDGFRα - CD31 − cells. EpCAM − MHCII + cells were then gated and PDPN + PDGFRβ - were classed at aaTEC2. Summary data represents mean ± SEM; each dot represents an individual biological replicate. Statistics for a–c were generated using two-tailed Mann–Whitney tests comparing within individual populations and for d using the Kruskal–Wallis test with Dunns correction.

Article Snippet: The antibodies used were rabbit anti-pan-cytokeratin (Dako, cat. no. Z0622), anti-K5 (BioLegend, cat. no. poly19055), rat anti-mouse K8/18 (Troma-1; Developmental Studies Hybridoma Bank), rabbit anti-K14 (Abcam, cat. no. EPR17350), rat anti-mouse AIRE (WEHI, clone 5H12), rabbit anti-human/mouse DCLK1 (LSBio, cat. no. LS-C100746) and biotinylated UEA1 lectin (Vector Labs, cat. no. B-1065).

Techniques: Flow Cytometry, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Nature Immunology

Article Title: Age-related epithelial defects limit thymic function and regeneration

doi: 10.1038/s41590-024-01915-9

Figure Lengend Snippet: a , Representative flow cytometry plots from 12–18-mo male and female Foxn1 nTnG mice at the indicated ages, gated on tdTom − GFP + cells. tdTom − GFP + EpCAM + cells were then assessed for expression of the conventional TEC markers UEA1 and Ly51. b , Claudin-3 and UEA1 expression on tdTom − GFP + EpCAM + cells in Foxn1 nTnG mice, and quantification of claudin-3 on UEA1 + mTEC and UEA1 − Ly51 − DN-TECs ( n = 4 biological replicates representing individual mice). c , Podoplanin (Pdpn) and PDGFRa expression on tdTom − GFP + EpCAM − cells ( n = 4 biological replicates representing individual mice). d , Number of GFP + EpCAM + UEA1 − Ly51 − Cldn3 + aaTEC1 and GFP + EpCAM − PDPN + PDGFRα − aaTEC2 cells in 2-mo ( n = 6) and 18-mo ( n = 4) mice. e , scRNA-seq was performed on CD45 − cells isolated from male and female 20-mo Foxn1 tdTom and age-matched WT mice, and integrated into the epithelial data described in Fig. . UMAP of 8,505 cells of the epithelial compartment in the integrated data showing the TEC annotated subsets (top) and overlaid expression of tdTomato (bottom). Scale represents log-transformed average expression of the tdTomato-WPRE element. f , RNA velocity on selected TEC populations in 2-mo (top) or 18-mo (bottom) mice. n 2mo = 1,989; n 18mo = 3,382. g , Vein plots describing the continuous transition of 18-mo early prog , mTEC1, mTEC prol and aaTEC subsets to their predicted descendants (represented by diagonal flows) and the dynamic relative frequencies (vein width on the y axis) of these TEC subsets in the thymus over the binned pseudotime. h , Expression of thymocyte markers Thy1 and Lck overlaid on the 18-mo spatial transcriptomics dataset. Outline represents thymocyte-poor area overlaid onto heatmap showing aaTEC1 or aaTEC2 signatures. i , Two representative images in 12–18-mo male and female Foxn1 nTnG mice showing tdTomato and GFP expression with HD-TEC areas highlighted, with few or no tdTomato + cells. Scale bar, 50 μm. j , Human tissue sections from a 50-year-old woman. Shown are consecutive sections with H&E, cytokeratin or CD1a staining. k , aaTEC1 and aaTEC2 gene signatures (top 20 marker genes from our mouse data converted to human orthologs; Supplementary Fig. and Supplementary Table ) were overlaid on human thymic epithelial cells ( n TEC = 40,144) from single-cell sequencing datasets generated and published elsewhere , , . Summary data represents mean ± s.e.m. and each dot represents an individual biological replicate. Statistics were generated ( b – d ) using a two-tailed Mann–Whitney test.

Article Snippet: The antibodies used were rabbit anti-pan-cytokeratin (Dako, cat. no. Z0622), anti-K5 (BioLegend, cat. no. poly19055), rat anti-mouse K8/18 (Troma-1; Developmental Studies Hybridoma Bank), rabbit anti-K14 (Abcam, cat. no. EPR17350), rat anti-mouse AIRE (WEHI, clone 5H12), rabbit anti-human/mouse DCLK1 (LSBio, cat. no. LS-C100746) and biotinylated UEA1 lectin (Vector Labs, cat. no. B-1065).

Techniques: Flow Cytometry, Expressing, Isolation, Transformation Assay, Staining, Marker, Sequencing, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Nature Immunology

Article Title: Age-related epithelial defects limit thymic function and regeneration

doi: 10.1038/s41590-024-01915-9

Figure Lengend Snippet: a , 3D reconstruction and representative images of high-density TEC region from 12-mo Foxn1 nTnG mice stained with DCLK1 or UEA1 to highlight tuft cells and M-like cells, respectively. b , Flow cytometry plots showing proportion of selected mimetic cells (tuft, corneocyte and M-cells) in 2-mo (n = 6) and 18-mo (n = 8) Foxn1 nTnG mice. Mimetic cells were first gated on EpCAM + GFP + cells, then mTECs (UEA1 hi Ly51 lo ) were assessed for the mimetic cell markers DCLK1 (tuft cells), GP2 (microfold cells), and Ly6D (corneocytes). Bar graph shows quantification of mimetic cell numbers. c , Flow cytometry plots showing mimetic cell frequency in 18-mo Foxn1 nTnG mice (n = 8) gated on EpCAM + GFP + UEA1 − Ly51 − DN-TECs. Bar graph shows quantification of mimetic cells comparing mTECs (as in Extended Data Fig. 7b) and DN-TECs. d , Aire and Foxn1 expression in TEC subsets. e , Representative confocal images of thymic sections from 12-mo Foxn1 nTnG mice stained with anti-AIRE, with the medulla or high-density TECs highlighted. Scale bar: 50μm. Summary data represents mean ± SEM; each dot represents an individual biological replicate. Statistics for b-c were generated using two-tailed Mann–Whitney tests comparing within individual mimetic cell subsets.

Article Snippet: The antibodies used were rabbit anti-pan-cytokeratin (Dako, cat. no. Z0622), anti-K5 (BioLegend, cat. no. poly19055), rat anti-mouse K8/18 (Troma-1; Developmental Studies Hybridoma Bank), rabbit anti-K14 (Abcam, cat. no. EPR17350), rat anti-mouse AIRE (WEHI, clone 5H12), rabbit anti-human/mouse DCLK1 (LSBio, cat. no. LS-C100746) and biotinylated UEA1 lectin (Vector Labs, cat. no. B-1065).

Techniques: Staining, Flow Cytometry, Expressing, Generated, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Neonatal microbiota colonization drives maturation of primary and secondary goblet cell mediated protection in the pre-weaning colon

doi: 10.1101/2024.07.03.601781

Figure Lengend Snippet: A) Ex vivo mucus growth in adult GF and ConvR mouse colon after stimulation with bacterial MAMPs. B) Ex vivo mucus growth dose-response to P3CSK4 in adult GF and ConvR mouse colon. C) Ex vivo mucus growth in adult GF and ConvR mouse colon stimulated with P3CSK4 in the presence or absence of senGC activation inhibitors. D) AB/PAS stained tissue sections from ex vivo experiments illustrated in (A). Emptied upper crypt GCs (red arrows) and lower crypt cavitation (yellow arrows) indicated. E) Whole mount confocal imaging of adult GF mouse colon treated with fluorescent Dextran tracer. Images show x/y-axis (upper panel) and x/z-axis (lower panel) cross-sections illustrating Dextran uptake by an upper crypt GC (purple arrow). F) Ex vivo mucus growth in neonatal (P3, 5, 15) and post weaning (P33) rat colon stimulated with P3CSK4 in the presence or absence of Dynasore inhibitor. G) Standardized expression of genes (columns) encoding known and predicted secreted proteins upregulated in mucus from P9-adult compared to P1-P7 rats (see ) in GC subpopulations (rows) identified by scRNA-seq. “Secretion” row indicates evidence of secretion determined by prior annotation or in silico prediction of classical or non-classical secretion by SecretomeP. H) Quantification of the frequency of Tgm3 expressing GCs as a proportion of the total GC population in neonatal (P3, P9, P14, P19) and post-weaning (P24) colonic tissue sections from ConvR mice. I) Confocal micrographs of representative tissue sections from P3 and P14 ConvR mice stained for Tgm3 (green) or the GC-binding lectins WGA (grey) and UEA1 (red). An individual GC from each image is indicated (yellow dashed line). Data represents n=3-5 (A-F, H-I)) animals per group, as indicated. All data is pooled from at least 2 independent litters or experiments. All histograms show median and interquartile range. Statistical comparisons between groups by 2-way ANOVA and Fishers LSD (A, C, F) or Kruskall Wallis and uncorrected Dunn’s test (H); p<0.05 (*), <0.01 (**) <0.001 (***), <0.0001 (****). Image scale bars are 50µm (D, I) or 20µm (E). # note: ConvR data displayed in A and B is reproduced from our previous publication and is shown for illustrative purposes only.

Article Snippet: Lastly, slides were washed with PBS and counterstained with a Hoechst-34580 DNA dye (5 μg/mL; Merck) in some cases supplemented with combinations of UEA1 Atto 488-conjugated lectin (10 µg/mL; Merck), UEA1 DyLight647-conjugated lectin (10 µg/mL; Vectorlabs) or WGA Alexa 555-conjugated lectin (10 µg/mL; ThermoFisher) for 15 min.

Techniques: Ex Vivo, Activation Assay, Staining, Imaging, Expressing, In Silico, Binding Assay

Journal: bioRxiv

Article Title: Neonatal microbiota colonization drives maturation of primary and secondary goblet cell mediated protection in the pre-weaning colon

doi: 10.1101/2024.07.03.601781

Figure Lengend Snippet: A) Schematic of the senGC activation pathway highlighting known (black) and putative (red) pathway genes. B) Expression of known and putative senGC genes in FACS isolated colonic GCs and colonocytes determined by DESeq2 analysis of bulk RNA sequencing data. C) Comparison of gene expression ratios between P22 ConvR:GF mice and adult 3-week ConvD:GF mice quantified by DESeq2 analysis of bulk colonic RNA sequencing data. Genes significantly up (red) or down (blue) regulated by microbiota exposure in both P22 and ConvD mice are indicated. D) Proportion of unique and shared genes significantly regulated by microbiota exposure in P22 ConvR and adult 3-week ConvD mice, based on data shown in (C). E) Comparison of microbiota-dependent expression of known and putative senGC activation pathway genes (A-B) in P22 ConvR and adult 3-week ConvD mice. Subset of data shown in (C). Genes not significantly regulated by microbiota in either group (grey), or genes regulated in either P22 ConvR (purple), adult ConvD (yellow) or both groups (teal) are indicated. F) Relative expression (compared to GF) of Duox2 (left) and Nox1 (right) genes in ConvD (brown) and B. fragilis monoassociated (blue) mice from 1-4 weeks colonization. Expression determined by qRT-PCR of colonic RNA, normalized to Gapdh and Rplp0 expression. G) Expression of Duox2 (left) and Nox1 (right) genes in postnatal ConvR (purple; P3-33) and GF (teal; P9-P33) determined by DESeq2 analysis of bulk colonic RNA sequencing data. H) Confocal micrographs of fixed colonic tissue sections from ConvR WT mice stained for Duox2 (left) and Nox1 (right) mRNA by in situ RNA hybridization and counterstained by Epcam (grey). Duox2 or Nox1 expressing crypt regions indicated (yellow arrows). I) Confocal micrographs showing upper crypt GCs in fixed colonic tissue sections from ConvR, GF and ConvD mice stained for Duox2 (red), mucus (UEA1; green), Actin (grey) and DNA (blue). Duox2 or Nox1 expressing crypt regions indicated (yellow arrows). J) Ex vivo mucus growth in Duox2 fl/fl and Duox2 ΔIEC colon tissue treated with carbachol (CCh), LPS or P3CSK4. K) Ex vivo mucus growth in WT colon tissue treated with P3CSK4 in the presence or absence of the Nox1 inhibitor ML171. Data represents n=2-5 animals per group, as indicated. All data is pooled from at least 2 independent experiments or litters. All histograms show median and interquartile range. Statistical comparisons between groups by DESeq2 (B, C, E), Kruskal Wallis and Dunn’s multiple comparison (F) or 2-way ANOVA and Fishers LSD (J, K); p<0.05 (*), <0.01 (**) <0.001 (***), <0.0001 (****). Scale bars are 50µm (H) or 10µm (I).

Article Snippet: Lastly, slides were washed with PBS and counterstained with a Hoechst-34580 DNA dye (5 μg/mL; Merck) in some cases supplemented with combinations of UEA1 Atto 488-conjugated lectin (10 µg/mL; Merck), UEA1 DyLight647-conjugated lectin (10 µg/mL; Vectorlabs) or WGA Alexa 555-conjugated lectin (10 µg/mL; ThermoFisher) for 15 min.

Techniques: Activation Assay, Expressing, Isolation, RNA Sequencing Assay, Comparison, Quantitative RT-PCR, Staining, In Situ, Hybridization, Ex Vivo