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rosetteseptm human ilc2 enrichment kit  (STEMCELL Technologies Inc)

 
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    STEMCELL Technologies Inc rosetteseptm human ilc2 enrichment kit
    IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), Alt (10μg) or PBS (25μl) over 1 week and culled 24h after the final dose. The frequency of <t>ILC2</t> (GFP+ CD45+ Linneg CD3-NKp46-CD127+CD90.2+KLRG1+CD25varIL-13+IL-5+) in the (A) airways (BAL fluid), (B) lung and (C) lung draining lymph nodes (mediastinal). Live viable precision cut lung slices of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow). Images of 1024μm x 1024μm field of view (FOV) were taken under a 20x objective using an inverted confocal microscope. (D) Images showing ILC2 (GFP+CD4-) CD4+ T cells (CD4+GFP-) location in PBS, rIL-33 and Alt treated mice, scale bar, 150 μm. (E) Number of ILC2 (GFP+CD4-) in lung sections per FOV taken under a 10x objective. (F) Schematic illustration of the lung depicting the anatomical location in the lung where precision cut lung slices were prepared. Representative images show two regions of the lung slice from a rIL-33 treated mouse showing distribution of ILC2 and CD4+ T cells, scale bar 150 μm. n = 4 mice per group (Mock(PBS)), n= 6 mice per group (Alt or rIL-33 treatment). Data representative of 4 experiments. *p < 0.05, **p < 0.01, ***p <0.001 and, **** p < 0.0001.
    Rosetteseptm Human Ilc2 Enrichment Kit, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rosetteseptm+human+ilc2+enrichment+kit/pmc06744282-666-7-12?v=STEMCELL+Technologies+Inc
    Average 90 stars, based on 1 article reviews
    rosetteseptm human ilc2 enrichment kit - by Bioz Stars, 2026-07
    90/100 stars

    Images

    1) Product Images from "Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans"

    Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

    Journal: Science immunology

    doi: 10.1126/sciimmunol.aav7638

    IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), Alt (10μg) or PBS (25μl) over 1 week and culled 24h after the final dose. The frequency of ILC2 (GFP+ CD45+ Linneg CD3-NKp46-CD127+CD90.2+KLRG1+CD25varIL-13+IL-5+) in the (A) airways (BAL fluid), (B) lung and (C) lung draining lymph nodes (mediastinal). Live viable precision cut lung slices of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow). Images of 1024μm x 1024μm field of view (FOV) were taken under a 20x objective using an inverted confocal microscope. (D) Images showing ILC2 (GFP+CD4-) CD4+ T cells (CD4+GFP-) location in PBS, rIL-33 and Alt treated mice, scale bar, 150 μm. (E) Number of ILC2 (GFP+CD4-) in lung sections per FOV taken under a 10x objective. (F) Schematic illustration of the lung depicting the anatomical location in the lung where precision cut lung slices were prepared. Representative images show two regions of the lung slice from a rIL-33 treated mouse showing distribution of ILC2 and CD4+ T cells, scale bar 150 μm. n = 4 mice per group (Mock(PBS)), n= 6 mice per group (Alt or rIL-33 treatment). Data representative of 4 experiments. *p < 0.05, **p < 0.01, ***p <0.001 and, **** p < 0.0001.
    Figure Legend Snippet: IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), Alt (10μg) or PBS (25μl) over 1 week and culled 24h after the final dose. The frequency of ILC2 (GFP+ CD45+ Linneg CD3-NKp46-CD127+CD90.2+KLRG1+CD25varIL-13+IL-5+) in the (A) airways (BAL fluid), (B) lung and (C) lung draining lymph nodes (mediastinal). Live viable precision cut lung slices of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow). Images of 1024μm x 1024μm field of view (FOV) were taken under a 20x objective using an inverted confocal microscope. (D) Images showing ILC2 (GFP+CD4-) CD4+ T cells (CD4+GFP-) location in PBS, rIL-33 and Alt treated mice, scale bar, 150 μm. (E) Number of ILC2 (GFP+CD4-) in lung sections per FOV taken under a 10x objective. (F) Schematic illustration of the lung depicting the anatomical location in the lung where precision cut lung slices were prepared. Representative images show two regions of the lung slice from a rIL-33 treated mouse showing distribution of ILC2 and CD4+ T cells, scale bar 150 μm. n = 4 mice per group (Mock(PBS)), n= 6 mice per group (Alt or rIL-33 treatment). Data representative of 4 experiments. *p < 0.05, **p < 0.01, ***p <0.001 and, **** p < 0.0001.

    Techniques Used: Staining, Microscopy

    IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), over 1 week and culled 24h after the final dose. Live viable precision cut lung slices (PCLS) of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), and time-lapse video taken (1024μm x 1024μm field of view (FOV), 45 min duration under a 20x objective using an inverted confocal microscope) (A) Static image depicting the location of ILC2 and CD4+ T cells, scale bar 100 μm. (B) Zoomed in section of the blood vessel in figure 2A, scale bar 20 μm. (C) High power images of boxed cells in figure 2B showing differences in pattern of cell movement (oscillatory vs amoeboid movement). ILC2 and CD4+ T cells dynamics were tracked and plotted as (D) individual tracks or (E) tracks commencing from centroid and overlaid. (F) Track speed, (G) track length and (H) track displacement were quantified. Representative images shown in (A-C) are from rIL-33 treated mice, where n = 6 mice per treatment (3 slices per mouse were imaged). For (F-H) in box and whiskers plots, each dot represents an individual cell. Data are representative from 4 experiments where n = 6 mice per treatment. *p < 0.05, **p < 0.01, ***p < 0.001 and, **** p < 0.0001.
    Figure Legend Snippet: IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), over 1 week and culled 24h after the final dose. Live viable precision cut lung slices (PCLS) of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), and time-lapse video taken (1024μm x 1024μm field of view (FOV), 45 min duration under a 20x objective using an inverted confocal microscope) (A) Static image depicting the location of ILC2 and CD4+ T cells, scale bar 100 μm. (B) Zoomed in section of the blood vessel in figure 2A, scale bar 20 μm. (C) High power images of boxed cells in figure 2B showing differences in pattern of cell movement (oscillatory vs amoeboid movement). ILC2 and CD4+ T cells dynamics were tracked and plotted as (D) individual tracks or (E) tracks commencing from centroid and overlaid. (F) Track speed, (G) track length and (H) track displacement were quantified. Representative images shown in (A-C) are from rIL-33 treated mice, where n = 6 mice per treatment (3 slices per mouse were imaged). For (F-H) in box and whiskers plots, each dot represents an individual cell. Data are representative from 4 experiments where n = 6 mice per treatment. *p < 0.05, **p < 0.01, ***p < 0.001 and, **** p < 0.0001.

    Techniques Used: Staining, Microscopy

    IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or Alt (10μg) over 1 week. Live precision cut lung slices were obtained and ILC2 dynamics were compared between the two and the differences were plotted as (A) individual tracks and (B) tracks commencing from centroid and overlaid. Differences in tracks between treatments were quantified as (C) track speed, (D) track length and (E) track displacement. Intravital microscopy (IVM) was performed in live IL-13eGFP mice after rIL-33 treatment (one 512μm x 512μm field of view (FOV) in a 1-hour-duration video). (F) Static images of different frames captured during the course of the video depicting amoeboid shape changes of ILC2 at separate time-points, scale bar 20 μm. n ≥ 4 mice per group. Data representative of 4 experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and, **** p < 0.0001. Quantifications from (A-E) are representative of 4 experiments, where n = 6 mice per treatment (3 slices per mouse were imaged). For (F) IVM images are representative of 6 individual IL-33 treated mice. *p < 0.05, **p < 0.01, ***p < 0.001, and, **** p < 0.0001.
    Figure Legend Snippet: IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or Alt (10μg) over 1 week. Live precision cut lung slices were obtained and ILC2 dynamics were compared between the two and the differences were plotted as (A) individual tracks and (B) tracks commencing from centroid and overlaid. Differences in tracks between treatments were quantified as (C) track speed, (D) track length and (E) track displacement. Intravital microscopy (IVM) was performed in live IL-13eGFP mice after rIL-33 treatment (one 512μm x 512μm field of view (FOV) in a 1-hour-duration video). (F) Static images of different frames captured during the course of the video depicting amoeboid shape changes of ILC2 at separate time-points, scale bar 20 μm. n ≥ 4 mice per group. Data representative of 4 experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and, **** p < 0.0001. Quantifications from (A-E) are representative of 4 experiments, where n = 6 mice per treatment (3 slices per mouse were imaged). For (F) IVM images are representative of 6 individual IL-33 treated mice. *p < 0.05, **p < 0.01, ***p < 0.001, and, **** p < 0.0001.

    Techniques Used: Intravital Microscopy

    IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose. (A) The percentage of murine ILC2 (CD45+LinnegNKp46-CD3-) expressing CCR1, CCR4 and CCR8. CCL8 levels in murine (B) BAL and (C) lung. (D) Location of CCL8 expression and ILC2 and (E) quantified CCL8 deposits in PCLS stained for CD31 (Magenta, the lung structure and blood vessels), CCL8 (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), images of 1024μm x 1024μm FOV, scale bar 150 μm. Human ILC2 lines were generated and migration to varying concentrations of (F) PGD2 and (G) CCL8 were determined. (H) Peak migratory responses of a human ILC2 cell line to IL-25, TGF-β, rIL-33, CCL8 and PGD2. IL13-eGFP mice treated with rIL-33 were also treated with 5μg purified anti-mouse CCR8 antibody i.p., rCCL8 i.n. or an isotype control and PCLS obtained and stained. (I) Localisation of ILC2 in live PCLS. (J) Number of ILC2 per FOV under 10x objective. Time-lapse imaging of 45 min duration was performed and ILC2 (K) track from centroid, (L) track length and (M) track speed and (N) track displacement were quantified. In box and whiskers graphs each data point represents an individual cell. Balb/c mice treated with rIL-33 were further treated with rCCL8, αCCR8 or Isotype control antibody. (O) Percentage of IL-13+IL-5+ILC2 (CD45+lin-NKp46-CD3-GATA-3+). (P) Representation Histogram of MFI of IL-13 and IL-5 and quantification of MFI for (Q) IL-13 and (R) IL-5 from GATA+ ILC2. For panels A-E n ≥ 4 mice per group. Data representative of 4 experiments. For panels F-H n = 3 individual donors. Data representative of 3 experiments. For panels I-R, n = 5 mice per group. Data representative of 2 experiments *p < 0.05, **p < 0.01, ***p < 0.001 and,**** p < 0.0001.
    Figure Legend Snippet: IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose. (A) The percentage of murine ILC2 (CD45+LinnegNKp46-CD3-) expressing CCR1, CCR4 and CCR8. CCL8 levels in murine (B) BAL and (C) lung. (D) Location of CCL8 expression and ILC2 and (E) quantified CCL8 deposits in PCLS stained for CD31 (Magenta, the lung structure and blood vessels), CCL8 (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), images of 1024μm x 1024μm FOV, scale bar 150 μm. Human ILC2 lines were generated and migration to varying concentrations of (F) PGD2 and (G) CCL8 were determined. (H) Peak migratory responses of a human ILC2 cell line to IL-25, TGF-β, rIL-33, CCL8 and PGD2. IL13-eGFP mice treated with rIL-33 were also treated with 5μg purified anti-mouse CCR8 antibody i.p., rCCL8 i.n. or an isotype control and PCLS obtained and stained. (I) Localisation of ILC2 in live PCLS. (J) Number of ILC2 per FOV under 10x objective. Time-lapse imaging of 45 min duration was performed and ILC2 (K) track from centroid, (L) track length and (M) track speed and (N) track displacement were quantified. In box and whiskers graphs each data point represents an individual cell. Balb/c mice treated with rIL-33 were further treated with rCCL8, αCCR8 or Isotype control antibody. (O) Percentage of IL-13+IL-5+ILC2 (CD45+lin-NKp46-CD3-GATA-3+). (P) Representation Histogram of MFI of IL-13 and IL-5 and quantification of MFI for (Q) IL-13 and (R) IL-5 from GATA+ ILC2. For panels A-E n ≥ 4 mice per group. Data representative of 4 experiments. For panels F-H n = 3 individual donors. Data representative of 3 experiments. For panels I-R, n = 5 mice per group. Data representative of 2 experiments *p < 0.05, **p < 0.01, ***p < 0.001 and,**** p < 0.0001.

    Techniques Used: Expressing, Staining, Generated, Migration, Purification, Control, Imaging

    Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV or serum free coating (control) for 24h. Cell movement was imaged via the JuLI imaging system and plotted as (A) individual tracks, (B) track speed dot plots and (C) track speed spider plot. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose PCLS obtained. (D) SHG imaging of PCLS revealing collagen fibres, representative maximum intensity projections, scale bar 50μm. (E) GLCM analysis of SHG imaging. (F) Images of Fibronectin expression and localisation. PCLS stained for CD31 (Magenta, the lung structure and blood vessels), Fibronectin (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow) and images of 1024μm x 1024μm field of view (FOV) were taken, scale bar 150μm. For panels A-C, n = 3 donors (in triplicate). Data representative of 3 experiments. For panels D-F n = 6 (in triplicate). *p < 0.05, **p < 0.01, ***p < 0.001 and,****, ****P < 0.0001.
    Figure Legend Snippet: Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV or serum free coating (control) for 24h. Cell movement was imaged via the JuLI imaging system and plotted as (A) individual tracks, (B) track speed dot plots and (C) track speed spider plot. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose PCLS obtained. (D) SHG imaging of PCLS revealing collagen fibres, representative maximum intensity projections, scale bar 50μm. (E) GLCM analysis of SHG imaging. (F) Images of Fibronectin expression and localisation. PCLS stained for CD31 (Magenta, the lung structure and blood vessels), Fibronectin (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow) and images of 1024μm x 1024μm field of view (FOV) were taken, scale bar 150μm. For panels A-C, n = 3 donors (in triplicate). Data representative of 3 experiments. For panels D-F n = 6 (in triplicate). *p < 0.05, **p < 0.01, ***p < 0.001 and,****, ****P < 0.0001.

    Techniques Used: Control, Imaging, Expressing, Staining

    Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV or serum free coating (control) and imaged after 12 hours. (A) Bright field images depicting change in shape. (B) Actin remodelling following staining with Phalloidin (green) and DAPI (cyan) and imaging using Airyscan detection (maximum intensity projections), scale bar 5μm. (C) Cell area. (D) Cell perimeter. n = 3 donors. Data representative of 2 experiments. *** P < 0.001.
    Figure Legend Snippet: Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV or serum free coating (control) and imaged after 12 hours. (A) Bright field images depicting change in shape. (B) Actin remodelling following staining with Phalloidin (green) and DAPI (cyan) and imaging using Airyscan detection (maximum intensity projections), scale bar 5μm. (C) Cell area. (D) Cell perimeter. n = 3 donors. Data representative of 2 experiments. *** P < 0.001.

    Techniques Used: Control, Staining, Imaging

    IL13-eGFP mice treated with rIL-33 were further treated with (BAPN) along with controls were culled 24 hours after the final dose. ILC2 dynamics from live PCLS were plotted as either (A) individual tracks or (B) tracks commencing from centroid and overlaid. Differences in tracks between treatments were quantified as (C) track speed, (D) track length and (E) track displacement. Total ILC2 () in (F) Lungs, (G) BAL and (H) Blood were enumerated. For panels A-E n ≥ 4 mice per group. Data representative of 4 experiments. For panels F-H n = 6 mice per group. Data representative of 2 experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
    Figure Legend Snippet: IL13-eGFP mice treated with rIL-33 were further treated with (BAPN) along with controls were culled 24 hours after the final dose. ILC2 dynamics from live PCLS were plotted as either (A) individual tracks or (B) tracks commencing from centroid and overlaid. Differences in tracks between treatments were quantified as (C) track speed, (D) track length and (E) track displacement. Total ILC2 () in (F) Lungs, (G) BAL and (H) Blood were enumerated. For panels A-E n ≥ 4 mice per group. Data representative of 4 experiments. For panels F-H n = 6 mice per group. Data representative of 2 experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

    Techniques Used:



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    rosetteseptm human ilc2 enrichment kit  (STEMCELL Technologies Inc)
    90
    STEMCELL Technologies Inc rosetteseptm human ilc2 enrichment kit
    IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), Alt (10μg) or PBS (25μl) over 1 week and culled 24h after the final dose. The frequency of <t>ILC2</t> (GFP+ CD45+ Linneg CD3-NKp46-CD127+CD90.2+KLRG1+CD25varIL-13+IL-5+) in the (A) airways (BAL fluid), (B) lung and (C) lung draining lymph nodes (mediastinal). Live viable precision cut lung slices of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow). Images of 1024μm x 1024μm field of view (FOV) were taken under a 20x objective using an inverted confocal microscope. (D) Images showing ILC2 (GFP+CD4-) CD4+ T cells (CD4+GFP-) location in PBS, rIL-33 and Alt treated mice, scale bar, 150 μm. (E) Number of ILC2 (GFP+CD4-) in lung sections per FOV taken under a 10x objective. (F) Schematic illustration of the lung depicting the anatomical location in the lung where precision cut lung slices were prepared. Representative images show two regions of the lung slice from a rIL-33 treated mouse showing distribution of ILC2 and CD4+ T cells, scale bar 150 μm. n = 4 mice per group (Mock(PBS)), n= 6 mice per group (Alt or rIL-33 treatment). Data representative of 4 experiments. *p < 0.05, **p < 0.01, ***p <0.001 and, **** p < 0.0001.
    Rosetteseptm Human Ilc2 Enrichment Kit, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rosetteseptm+human+ilc2+enrichment+kit/pmc06744282-666-7-12?v=STEMCELL+Technologies+Inc
    Average 90 stars, based on 1 article reviews
    rosetteseptm human ilc2 enrichment kit - by Bioz Stars, 2026-07
    90/100 stars
    		  Buy from Supplier

    Image Search Results


    IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), Alt (10μg) or PBS (25μl) over 1 week and culled 24h after the final dose. The frequency of ILC2 (GFP+ CD45+ Linneg CD3-NKp46-CD127+CD90.2+KLRG1+CD25varIL-13+IL-5+) in the (A) airways (BAL fluid), (B) lung and (C) lung draining lymph nodes (mediastinal). Live viable precision cut lung slices of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow). Images of 1024μm x 1024μm field of view (FOV) were taken under a 20x objective using an inverted confocal microscope. (D) Images showing ILC2 (GFP+CD4-) CD4+ T cells (CD4+GFP-) location in PBS, rIL-33 and Alt treated mice, scale bar, 150 μm. (E) Number of ILC2 (GFP+CD4-) in lung sections per FOV taken under a 10x objective. (F) Schematic illustration of the lung depicting the anatomical location in the lung where precision cut lung slices were prepared. Representative images show two regions of the lung slice from a rIL-33 treated mouse showing distribution of ILC2 and CD4+ T cells, scale bar 150 μm. n = 4 mice per group (Mock(PBS)), n= 6 mice per group (Alt or rIL-33 treatment). Data representative of 4 experiments. *p < 0.05, **p < 0.01, ***p <0.001 and, **** p < 0.0001.

    Journal: Science immunology

    Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

    doi: 10.1126/sciimmunol.aav7638

    Figure Lengend Snippet: IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), Alt (10μg) or PBS (25μl) over 1 week and culled 24h after the final dose. The frequency of ILC2 (GFP+ CD45+ Linneg CD3-NKp46-CD127+CD90.2+KLRG1+CD25varIL-13+IL-5+) in the (A) airways (BAL fluid), (B) lung and (C) lung draining lymph nodes (mediastinal). Live viable precision cut lung slices of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow). Images of 1024μm x 1024μm field of view (FOV) were taken under a 20x objective using an inverted confocal microscope. (D) Images showing ILC2 (GFP+CD4-) CD4+ T cells (CD4+GFP-) location in PBS, rIL-33 and Alt treated mice, scale bar, 150 μm. (E) Number of ILC2 (GFP+CD4-) in lung sections per FOV taken under a 10x objective. (F) Schematic illustration of the lung depicting the anatomical location in the lung where precision cut lung slices were prepared. Representative images show two regions of the lung slice from a rIL-33 treated mouse showing distribution of ILC2 and CD4+ T cells, scale bar 150 μm. n = 4 mice per group (Mock(PBS)), n= 6 mice per group (Alt or rIL-33 treatment). Data representative of 4 experiments. *p < 0.05, **p < 0.01, ***p <0.001 and, **** p < 0.0001.

    Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

    Techniques: Staining, Microscopy

    IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), over 1 week and culled 24h after the final dose. Live viable precision cut lung slices (PCLS) of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), and time-lapse video taken (1024μm x 1024μm field of view (FOV), 45 min duration under a 20x objective using an inverted confocal microscope) (A) Static image depicting the location of ILC2 and CD4+ T cells, scale bar 100 μm. (B) Zoomed in section of the blood vessel in figure 2A, scale bar 20 μm. (C) High power images of boxed cells in figure 2B showing differences in pattern of cell movement (oscillatory vs amoeboid movement). ILC2 and CD4+ T cells dynamics were tracked and plotted as (D) individual tracks or (E) tracks commencing from centroid and overlaid. (F) Track speed, (G) track length and (H) track displacement were quantified. Representative images shown in (A-C) are from rIL-33 treated mice, where n = 6 mice per treatment (3 slices per mouse were imaged). For (F-H) in box and whiskers plots, each dot represents an individual cell. Data are representative from 4 experiments where n = 6 mice per treatment. *p < 0.05, **p < 0.01, ***p < 0.001 and, **** p < 0.0001.

    Journal: Science immunology

    Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

    doi: 10.1126/sciimmunol.aav7638

    Figure Lengend Snippet: IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), over 1 week and culled 24h after the final dose. Live viable precision cut lung slices (PCLS) of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), and time-lapse video taken (1024μm x 1024μm field of view (FOV), 45 min duration under a 20x objective using an inverted confocal microscope) (A) Static image depicting the location of ILC2 and CD4+ T cells, scale bar 100 μm. (B) Zoomed in section of the blood vessel in figure 2A, scale bar 20 μm. (C) High power images of boxed cells in figure 2B showing differences in pattern of cell movement (oscillatory vs amoeboid movement). ILC2 and CD4+ T cells dynamics were tracked and plotted as (D) individual tracks or (E) tracks commencing from centroid and overlaid. (F) Track speed, (G) track length and (H) track displacement were quantified. Representative images shown in (A-C) are from rIL-33 treated mice, where n = 6 mice per treatment (3 slices per mouse were imaged). For (F-H) in box and whiskers plots, each dot represents an individual cell. Data are representative from 4 experiments where n = 6 mice per treatment. *p < 0.05, **p < 0.01, ***p < 0.001 and, **** p < 0.0001.

    Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

    Techniques: Staining, Microscopy

    IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or Alt (10μg) over 1 week. Live precision cut lung slices were obtained and ILC2 dynamics were compared between the two and the differences were plotted as (A) individual tracks and (B) tracks commencing from centroid and overlaid. Differences in tracks between treatments were quantified as (C) track speed, (D) track length and (E) track displacement. Intravital microscopy (IVM) was performed in live IL-13eGFP mice after rIL-33 treatment (one 512μm x 512μm field of view (FOV) in a 1-hour-duration video). (F) Static images of different frames captured during the course of the video depicting amoeboid shape changes of ILC2 at separate time-points, scale bar 20 μm. n ≥ 4 mice per group. Data representative of 4 experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and, **** p < 0.0001. Quantifications from (A-E) are representative of 4 experiments, where n = 6 mice per treatment (3 slices per mouse were imaged). For (F) IVM images are representative of 6 individual IL-33 treated mice. *p < 0.05, **p < 0.01, ***p < 0.001, and, **** p < 0.0001.

    Journal: Science immunology

    Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

    doi: 10.1126/sciimmunol.aav7638

    Figure Lengend Snippet: IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or Alt (10μg) over 1 week. Live precision cut lung slices were obtained and ILC2 dynamics were compared between the two and the differences were plotted as (A) individual tracks and (B) tracks commencing from centroid and overlaid. Differences in tracks between treatments were quantified as (C) track speed, (D) track length and (E) track displacement. Intravital microscopy (IVM) was performed in live IL-13eGFP mice after rIL-33 treatment (one 512μm x 512μm field of view (FOV) in a 1-hour-duration video). (F) Static images of different frames captured during the course of the video depicting amoeboid shape changes of ILC2 at separate time-points, scale bar 20 μm. n ≥ 4 mice per group. Data representative of 4 experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and, **** p < 0.0001. Quantifications from (A-E) are representative of 4 experiments, where n = 6 mice per treatment (3 slices per mouse were imaged). For (F) IVM images are representative of 6 individual IL-33 treated mice. *p < 0.05, **p < 0.01, ***p < 0.001, and, **** p < 0.0001.

    Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

    Techniques: Intravital Microscopy

    IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose. (A) The percentage of murine ILC2 (CD45+LinnegNKp46-CD3-) expressing CCR1, CCR4 and CCR8. CCL8 levels in murine (B) BAL and (C) lung. (D) Location of CCL8 expression and ILC2 and (E) quantified CCL8 deposits in PCLS stained for CD31 (Magenta, the lung structure and blood vessels), CCL8 (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), images of 1024μm x 1024μm FOV, scale bar 150 μm. Human ILC2 lines were generated and migration to varying concentrations of (F) PGD2 and (G) CCL8 were determined. (H) Peak migratory responses of a human ILC2 cell line to IL-25, TGF-β, rIL-33, CCL8 and PGD2. IL13-eGFP mice treated with rIL-33 were also treated with 5μg purified anti-mouse CCR8 antibody i.p., rCCL8 i.n. or an isotype control and PCLS obtained and stained. (I) Localisation of ILC2 in live PCLS. (J) Number of ILC2 per FOV under 10x objective. Time-lapse imaging of 45 min duration was performed and ILC2 (K) track from centroid, (L) track length and (M) track speed and (N) track displacement were quantified. In box and whiskers graphs each data point represents an individual cell. Balb/c mice treated with rIL-33 were further treated with rCCL8, αCCR8 or Isotype control antibody. (O) Percentage of IL-13+IL-5+ILC2 (CD45+lin-NKp46-CD3-GATA-3+). (P) Representation Histogram of MFI of IL-13 and IL-5 and quantification of MFI for (Q) IL-13 and (R) IL-5 from GATA+ ILC2. For panels A-E n ≥ 4 mice per group. Data representative of 4 experiments. For panels F-H n = 3 individual donors. Data representative of 3 experiments. For panels I-R, n = 5 mice per group. Data representative of 2 experiments *p < 0.05, **p < 0.01, ***p < 0.001 and,**** p < 0.0001.

    Journal: Science immunology

    Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

    doi: 10.1126/sciimmunol.aav7638

    Figure Lengend Snippet: IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose. (A) The percentage of murine ILC2 (CD45+LinnegNKp46-CD3-) expressing CCR1, CCR4 and CCR8. CCL8 levels in murine (B) BAL and (C) lung. (D) Location of CCL8 expression and ILC2 and (E) quantified CCL8 deposits in PCLS stained for CD31 (Magenta, the lung structure and blood vessels), CCL8 (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), images of 1024μm x 1024μm FOV, scale bar 150 μm. Human ILC2 lines were generated and migration to varying concentrations of (F) PGD2 and (G) CCL8 were determined. (H) Peak migratory responses of a human ILC2 cell line to IL-25, TGF-β, rIL-33, CCL8 and PGD2. IL13-eGFP mice treated with rIL-33 were also treated with 5μg purified anti-mouse CCR8 antibody i.p., rCCL8 i.n. or an isotype control and PCLS obtained and stained. (I) Localisation of ILC2 in live PCLS. (J) Number of ILC2 per FOV under 10x objective. Time-lapse imaging of 45 min duration was performed and ILC2 (K) track from centroid, (L) track length and (M) track speed and (N) track displacement were quantified. In box and whiskers graphs each data point represents an individual cell. Balb/c mice treated with rIL-33 were further treated with rCCL8, αCCR8 or Isotype control antibody. (O) Percentage of IL-13+IL-5+ILC2 (CD45+lin-NKp46-CD3-GATA-3+). (P) Representation Histogram of MFI of IL-13 and IL-5 and quantification of MFI for (Q) IL-13 and (R) IL-5 from GATA+ ILC2. For panels A-E n ≥ 4 mice per group. Data representative of 4 experiments. For panels F-H n = 3 individual donors. Data representative of 3 experiments. For panels I-R, n = 5 mice per group. Data representative of 2 experiments *p < 0.05, **p < 0.01, ***p < 0.001 and,**** p < 0.0001.

    Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

    Techniques: Expressing, Staining, Generated, Migration, Purification, Control, Imaging

    Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV or serum free coating (control) for 24h. Cell movement was imaged via the JuLI imaging system and plotted as (A) individual tracks, (B) track speed dot plots and (C) track speed spider plot. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose PCLS obtained. (D) SHG imaging of PCLS revealing collagen fibres, representative maximum intensity projections, scale bar 50μm. (E) GLCM analysis of SHG imaging. (F) Images of Fibronectin expression and localisation. PCLS stained for CD31 (Magenta, the lung structure and blood vessels), Fibronectin (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow) and images of 1024μm x 1024μm field of view (FOV) were taken, scale bar 150μm. For panels A-C, n = 3 donors (in triplicate). Data representative of 3 experiments. For panels D-F n = 6 (in triplicate). *p < 0.05, **p < 0.01, ***p < 0.001 and,****, ****P < 0.0001.

    Journal: Science immunology

    Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

    doi: 10.1126/sciimmunol.aav7638

    Figure Lengend Snippet: Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV or serum free coating (control) for 24h. Cell movement was imaged via the JuLI imaging system and plotted as (A) individual tracks, (B) track speed dot plots and (C) track speed spider plot. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose PCLS obtained. (D) SHG imaging of PCLS revealing collagen fibres, representative maximum intensity projections, scale bar 50μm. (E) GLCM analysis of SHG imaging. (F) Images of Fibronectin expression and localisation. PCLS stained for CD31 (Magenta, the lung structure and blood vessels), Fibronectin (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow) and images of 1024μm x 1024μm field of view (FOV) were taken, scale bar 150μm. For panels A-C, n = 3 donors (in triplicate). Data representative of 3 experiments. For panels D-F n = 6 (in triplicate). *p < 0.05, **p < 0.01, ***p < 0.001 and,****, ****P < 0.0001.

    Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

    Techniques: Control, Imaging, Expressing, Staining

    Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV or serum free coating (control) and imaged after 12 hours. (A) Bright field images depicting change in shape. (B) Actin remodelling following staining with Phalloidin (green) and DAPI (cyan) and imaging using Airyscan detection (maximum intensity projections), scale bar 5μm. (C) Cell area. (D) Cell perimeter. n = 3 donors. Data representative of 2 experiments. *** P < 0.001.

    Journal: Science immunology

    Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

    doi: 10.1126/sciimmunol.aav7638

    Figure Lengend Snippet: Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV or serum free coating (control) and imaged after 12 hours. (A) Bright field images depicting change in shape. (B) Actin remodelling following staining with Phalloidin (green) and DAPI (cyan) and imaging using Airyscan detection (maximum intensity projections), scale bar 5μm. (C) Cell area. (D) Cell perimeter. n = 3 donors. Data representative of 2 experiments. *** P < 0.001.

    Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

    Techniques: Control, Staining, Imaging

    IL13-eGFP mice treated with rIL-33 were further treated with (BAPN) along with controls were culled 24 hours after the final dose. ILC2 dynamics from live PCLS were plotted as either (A) individual tracks or (B) tracks commencing from centroid and overlaid. Differences in tracks between treatments were quantified as (C) track speed, (D) track length and (E) track displacement. Total ILC2 () in (F) Lungs, (G) BAL and (H) Blood were enumerated. For panels A-E n ≥ 4 mice per group. Data representative of 4 experiments. For panels F-H n = 6 mice per group. Data representative of 2 experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

    Journal: Science immunology

    Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

    doi: 10.1126/sciimmunol.aav7638

    Figure Lengend Snippet: IL13-eGFP mice treated with rIL-33 were further treated with (BAPN) along with controls were culled 24 hours after the final dose. ILC2 dynamics from live PCLS were plotted as either (A) individual tracks or (B) tracks commencing from centroid and overlaid. Differences in tracks between treatments were quantified as (C) track speed, (D) track length and (E) track displacement. Total ILC2 () in (F) Lungs, (G) BAL and (H) Blood were enumerated. For panels A-E n ≥ 4 mice per group. Data representative of 4 experiments. For panels F-H n = 6 mice per group. Data representative of 2 experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

    Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

    Techniques: