photon microscopy data  (Cell Signaling Technology Inc)

Journal: Theranostics

Article Title: Intravital imaging of interactions between iNKT and kupffer cells to clear free lipids during steatohepatitis

doi: 10.7150/thno.51369

Figure Lengend Snippet: Recruited iNKT cells form aggregated clusters and display distinct migratory patterns in early steatohepatitis . (A) Left: Frequencies of iNKT cells (GFP + CD1d - tetramers + ) among liver leukocytes of C57BL/6 mice (n = 6) fed MCS or MCD diet for 3 or 5 days, as evaluated by flow cytometry. Right: Quantification of recruited iNKT cells in mice livers fed MCS or MCD diet for 3 or 5 days (n = 6). (B) Cxcr6 Gfp/+ mice fed MCS or MCD diet for 3 or 5 days were subjected to hepatic intravital microscopy. Representative images were obtained from ≥ 3 independent experiments to examine iNKT cell response (green). The sinusoidal endothelium was labeled using Alexa Fluor 647-conjugated anti-platelet endothelial cell adhesion molecule (PECAM-1) antibodies (red). The image in the square with a gray outline is partially enlarged. iNKT cells were divided into three states: normal state, outside the cluster, and inside the cluster. Scale bar, 100 µm. iNKT cell clusters are indicated with a white dashed line. (C) Overlay of three states iNKT cell migratory tracks plotted after aligning their starting positions. Each plot was collected from 20 iNKT cells from three different Cxcr6 Gfp/+ mice, and each scan was imaged for 10 min. (D) Scatter plots of the velocity, distance, and migratory displacement of migrating iNKT cells in the liver of Cxcr6 Gfp/+ mice (n = 3) fed MCS or MCD diet for 3 days. Scatter plots or displacement curves of the arrested coefficient, Confinement ratio, and mean displacement (µm) versus the square root of the time (min 1/2 ) of migrating iNKT cells in the liver of Cxcr6 Gfp/+ mice (n = 3) fed an MCS or MCD diet for 3 days. Fifty migrating iNKT cells from three independent experiments were pooled. Data represent mean relative expression ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 using two-tailed unpaired student's t-test.

Article Snippet: Intravital single-photon laser scanning microscopy (SPLSM) data were analyzed using Imaris 7.4.2 (Bitplane).

Techniques: Flow Cytometry, Intravital Microscopy, Labeling, Expressing, Two Tailed Test

Journal: Theranostics

Article Title: Intravital imaging of interactions between iNKT and kupffer cells to clear free lipids during steatohepatitis

doi: 10.7150/thno.51369

Figure Lengend Snippet: Kupffer and iNKT cells cluster together and interact dynamically, and aggregation properties of Kupffer cells depend on the aggregation of iNKT cells. (A) Left: Representative Z-Stack fluorescence co-localization images (X = 100 µm, Y = 100 µm, Z = 25 µm) were obtained from ≥ 3 experiments performed to examine the interactions between iNKT cells and Kupffer cells in different states. Three-dimensionally rendered blue was used to indicate iNKT and green for Kupffer cell (Fuchsia, labeled with Alexa Fluor® 647-conjugated anti-mouse F4/80 antibodies) interactions. Right: Quantification of the co-located area of iNKT and Kupffer cell interactions in three states (n = 20). (B) Left: Cxcr6 Gfp/+ mice fed MCS or MCD diet were subjected to hepatic intravital microscopy. Representative Z-Stack images (X = 425 µm, Y = 425 µm, Z = 25 µm) were obtained from ≥ 3 experiments performed to examine the interactions between iNKT cells (green) and Kupffer cells (Fuchsia, labeled with Alexa Fluor® 647-conjugated anti-mouse F4/80 antibodies). Right: Quantification of the number of iNKT cell clusters and Kupffer cell clusters in untreated or MCD-fed Cxcr6 Gfp/+ mice (n = 5). (C) Left: Representative images were obtained from 3 independent experiments to examine the formation ability of cell clusters composed of iNKT cells (green)and Kupffer cells (Fuchsia, labeled with Alexa Fluor® 647-conjugated anti-mouse F4/80 antibodies). WT (Cxcr6 Gfp/+ mice) or iNKT cell-deficient mice (CD1d -/- mice, Jα18 -/- mice) treated with clodronate liposomes (CLL) or PBS fed MCD diet for 3, 5, and 7 days. Scale bar, 100 µm. Right: Quantification of the number of iNKT cell clusters and Kupffer cell clusters in WT (Cxcr6 Gfp/+ mice) or iNKT cell-deficient mice (CD1d -/- mice, Jα18 -/- mice) (n = 5). Data represent mean relative expression ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 using two-tailed unpaired student's t-test.

Article Snippet: Intravital single-photon laser scanning microscopy (SPLSM) data were analyzed using Imaris 7.4.2 (Bitplane).

Techniques: Fluorescence, Labeling, Intravital Microscopy, Liposomes, Expressing, Two Tailed Test

Journal: Theranostics

Article Title: Intravital imaging of interactions between iNKT and kupffer cells to clear free lipids during steatohepatitis

doi: 10.7150/thno.51369

Figure Lengend Snippet: Aggregated iNKT cells have the lipid phagocytosis ability. (A) Cxcr6 Gfp/+ mice fed MCD diet for 3 days were subjected to hepatic intravital microscopy. Time-lapse images show snapshots of a green iNKT cell (green) approaching a triglyceride (TG) drop (stained with Nile red and indicated with a white circle) and eventually engulfing it. See also . (B) Left: Representative 3D images from ≥ 3 independent experiments showing aggregated iNKT cells (green) and TG drops (red, stained with Nile red) in the liver of Cxcr6 Gfp/+ mice fed MCD diet. 3D image dimensions (µm): X = 54 µm, Y = 54 µm, Z = 25 µm, where the XY plane is perpendicular to the objective lens. Representative 3D intersection images and single-cell cutting (after 3D surface rendering) show the hepatic iNKT cell (green) ability to engulf lipid drops (red). Right: Quantification of iNKT cell number with TG drops and volume of lipid drops engulfed in the cytoplasm outside or inside the iNKT cell cluster in the liver (n = 20). (C) Left: Representative images were obtained from ≥ 3 experiments to examine lipids (red, Nile red labeled lipid) in clustered or non-aggregated Kupffer cells (purple, F4/80 antibody-labeled Kupffer cells). 3D images of Kupffer cells and lipids obtained by Z-stacking were scanned. Kupffer cells (3D rendering) in the cell cluster show co-localization with the lipid. X = 142 µm, Y = 142 µm, Z = 30 µm, where the XY plane is perpendicular to the objective lens. Right: Quantification of volume of lipid drops engulfed by Kupffer cells outside or inside the clusters in the liver (n = 15). (D) Left: Representative images were obtained from ≥ 3 experiments to examine lipids (red, Nile red labeled lipid) in iNKT cells (green) and Kupffer cells (purple, F4/80 antibody-labeled Kupffer cells). 3D images of iNKT cells, Kupffer cells, and lipids obtained by Z-stacking were scanned. iNKT cells (co-localized in red, 3D rendering) and Kupffer cells (colocalized in aqua blue, 3D rendering) in the cell cluster show co-localization with the lipid. X = 108 µm, Y = 108 µm, Z = 30 µm, where the XY plane is perpendicular to the objective lens. Right: Quantification of the volume of engulfed lipid drops in aggregated iNKT cells or Kupffer cells in Cxcr6 Gfp/+ mice fed MCD diet (n = 15). Data represent mean relative expression ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 using two-tailed unpaired student's t-test.

Article Snippet: Intravital single-photon laser scanning microscopy (SPLSM) data were analyzed using Imaris 7.4.2 (Bitplane).

Techniques: Intravital Microscopy, Staining, Labeling, Expressing, Two Tailed Test