Journal: Scientific Reports

Article Title: Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

doi: 10.1038/s41598-017-15212-z

Figure Lengend Snippet: Human retina tissue expression and iPS-RPE cell expression patterns of CL-11. ( a ) Representative confocal image showing CL-11 expression on permeabilised (left panel) and non-permeabilised (middle panel) human retinal sections. The sections shown are taken from the peripheral retina and from the central retina outside the macula. Control sections were incubated with no primary antibody (right panel). CL-11 (green), nuclei (blue) and autofluorescence (red) are shown. Scale bars, 25 μm. CL-11 staining without DAPI is shown on the right side of peripheral retina and central retina panels. High magnification images of area indicated in the white box are shown in the bottom panel. Scale bar, 10 μm. ( b ) Representative confocal microscopic image showing differentiated iPS-RPE cells morphology (left panel) and CL-11 staining on permeabilised human iPS-RPE cells (middle and right panel). CL-11 (red), nuclei (blue) and phalloidin (aqua) are shown. Scale bars, 25 μm ( c ) Flow cytometry histograms showing CL-11 expression on permeabilised iPS-RPE cells (intracellular) versus non-permeabilised cells (surface). Cells stained with secondary antibody alone were used as control.

Article Snippet: Following 24 hours, the cells were washed in PBS and fixed in 4% paraformaldehyde (but not permeabilised) for 1 hour at room temperature. iPS-RPE cells were then stained for CL-11 by immunofluorescence using either rabbit polyclonal anti-CL-11 antibody (1:100, Abbexa) or a mouse monoclonal IgG anti-human CL-11 produced in house.

Techniques: Expressing, Incubation, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

doi: 10.1038/s41598-017-15212-z

Figure Lengend Snippet: CL-11 binding to hypoxia-stressed iPS-RPE cells. ( a ) Representative Western blot under reducing conditions showing CL-11 expression in iPS-RPE cells under normal and hypoxic conditions. HSP90 was used as a loading control. Full-length blots are presented in Supplemental Fig. . ( b ) Relative density analysis of CL-11 in iPS-RPE cells following different treatments (N = 3 independent experiments). ( c ) Detection of both non-reduced (100 kDa) and reduced states (34 kDa) of CL-11 in the culture supernatants of iPS-RPE cells following or not hypoxic stress by Western blotting. ( d ) Representative immunofluorescence images showing CL-11 binding to the surface of hypoxic-stressed iPS- RPE cells compared to non-stressed cells. Scale bars, 25 μm. ( e ) Quantification of CL-11 binding in normal and hypoxic iPS-RPE cells using ImageJ software. CL-11 (red), nuclei (blue) and phalloidin (aqua) are shown. N = 3 independent experiments, * **P < 0,0001, t -test. ( f ) Confocal images of CL-11 (red) and L-fucose (green) showing co-expression of CL-11 and L-fucose (orange) on hypoxic–iPS-RPE cells treated or untreated with fucosidase. Scale bars, 50 μm. ( g ) Quantification of L-fucose expression and ( h ) CL-11 binding to hypoxia-stressed cells treated or untreated with fucosidase using ImageJ software. N = 4 independent experiments.

Article Snippet: Following 24 hours, the cells were washed in PBS and fixed in 4% paraformaldehyde (but not permeabilised) for 1 hour at room temperature. iPS-RPE cells were then stained for CL-11 by immunofluorescence using either rabbit polyclonal anti-CL-11 antibody (1:100, Abbexa) or a mouse monoclonal IgG anti-human CL-11 produced in house.

Techniques: Binding Assay, Western Blot, Expressing, Immunofluorescence, Software

Journal: Scientific Reports

Article Title: Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

doi: 10.1038/s41598-017-15212-z

Figure Lengend Snippet: C3d deposition and Mac assembly on hypoxia-stressed iPS-RPE cells. ( a ) Representative Western blot showing C3 protein expression in iPS-RPE cells under normal and hypoxic conditions. Actin was used as a loading control. Full-length blots are presented in Supplemental Fig. . ( b ) Representative immunofluorescence images showing CL-11 (red) and C3d (green) staining on hypoxia-stressed iPS- RPE cells compared to non-stressed cells. Nuclei (blue) and phalloidin (grey) are shown. Scale bars, 25 μm. Quantification of CL-11 ( c ) and C3d ( d ) expression following or not hypoxia stress using ImageJ software. N = 3 independent experiments; n = 15 images analysed * **P < 0,0001, t -test. ( e ) CL-11 and C3d co-localization was quantified by calculating Mander’s co-localization coefficients using JACoP plug-in and ImageJ software. ( f ) Representative confocal images of CL-11 (green), MAC (red) nuclei (blue) and phalloidin (aqua) showing co-expression of CL-11 and MAC on hypoxic iPS-RPE compared to non-stressed cells. Scale bars, 25 μm. High magnification image (white box) showing co-localization of CL-11 and MAC. Scale bars, 10 μm. ( g ) CL-11 and MAC co-localization was quantified by calculating Mander’s co-localization coefficients using JACoP plug-in and ImageJ software. N = 3 independent experiments.

Article Snippet: Following 24 hours, the cells were washed in PBS and fixed in 4% paraformaldehyde (but not permeabilised) for 1 hour at room temperature. iPS-RPE cells were then stained for CL-11 by immunofluorescence using either rabbit polyclonal anti-CL-11 antibody (1:100, Abbexa) or a mouse monoclonal IgG anti-human CL-11 produced in house.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Software

Journal: Scientific Reports

Article Title: Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

doi: 10.1038/s41598-017-15212-z

Figure Lengend Snippet: CL-11 and MASP-2 interaction on hypoxia-stressed iPS-RPE cells. ( a ) Representative Western blot showing MASP-2 protein expression in iPS-RPE cells under normal and hypoxic conditions. HSP90 was used as a loading control. Full-length blots are presented in Supplemental Fig. . ( b ) Relative density analysis of MASP-2 in iPS-RPE cells under different treatments. N = 3 independent experiments. ( c ) Representative immunofluorescence images showing MASP-2 staining on hypoxia-stressed iPS- RPE cells compared to non-stressed cells. MASP-2 (red) and phalloidin (aqua) are shown on the left panels and MASP-2 (red) alone is shown on the right panels. Scale bars, 25 μm. ( d ) Representative immunofluorescence images showing co-expression of CL-11 and MASP-2 (orange) on the surface of hypoxia-stressed iPS- RPE cells MASP-2 (red), CL-11 (green) nuclei (blue) and phalloidin (grey) are shown. Scale bars, 25 μm. ( e ) CL-11 and MASP-2 co-localization was quantified by calculating Mander’s co-localization coefficients using JACoP plug-in and ImageJ software.

Article Snippet: Following 24 hours, the cells were washed in PBS and fixed in 4% paraformaldehyde (but not permeabilised) for 1 hour at room temperature. iPS-RPE cells were then stained for CL-11 by immunofluorescence using either rabbit polyclonal anti-CL-11 antibody (1:100, Abbexa) or a mouse monoclonal IgG anti-human CL-11 produced in house.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Software