Journal: Scientific Reports

Article Title: Laminin-511-E8 promotes efficient in vitro expansion of human limbal melanocytes

doi: 10.1038/s41598-020-68120-0

Figure Lengend Snippet: Effect of laminin isoforms on limbal melanocyte function in vitro. ( A ) Effect of laminin (LN) 521, 511-E8, 421, 332, 211, and 111 on cell adhesion compared with control 30 min after seeding. Data are expressed as means ± SEM (n = 4) (left). Phase contrast images of limbal melanocytes after 30 min of adhesion to LN isoforms (right); magnification × 100. ( B ) Functional blocking of integrin-mediated cell adhesion to LN521, LN511-E8, and LN332 was tested using neutralizing antibodies against integrin α3β1 and α6β1 30 min after seeding. Data are expressed as means ± SEM (n = 4). ( C ) The effect of LN isoforms on cell migration was analyzed in two well-culture inserts with a defined cell-free gap and measurement of gap closure 12 and 24 h after removal of the culture inserts. Data are expressed as means ± SEM (n = 3). ( D ) The effect of LN isoforms on cell proliferation was analyzed by BrdU incorporation (left) and immunocytochemical Ki67 expression (right) 72 h after incubation. Data are expressed as means ± SEM (n = 4). Immunocytochemistry shows increased nuclear expression of Ki67 (magenta) in Melan-A + melanocytes (green) grown on LN-511-E8 compared to control; nuclei are counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue). ( E ) The additional effect of various growth factors on proliferation of melanocytes cultured on LN-511-E8 coated plates was analyzed by BrdU incorporation (left) and immunocytochemical Ki67 expression (right) 48 h after incubation. Data are expressed as means ± SEM (n = 4). Immunocytochemistry shows increased nuclear expression of Ki67 (magenta) in Melan-A + melanocytes (green) cultured in the presence of bFGF; nuclear counterstained with DAPI (blue). (bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; ET-1, endothelin 1; GM-CSF, granulocyte macrophage colony stimulating factor; HGF, hepatocyte growth factor; LIF, leukemia inhibitory factor; α-MSH, α-melanocyte stimulating hormone; SCF, stem cell factor; TGF-ß, transforming growth factor ß1; TPA, 12- O -tetradecanoylphorbol 13-acetate; Y-27632, ROCK inhibitor). ( F ) Phase contrast images (top) of melanocytes cultured on LN-511-E8 and tissue culture plastic (control); magnification × 100. Qunatification of cellular melanin content (bottom) in melanocytes cultured on LN-511-E8 and tissue culture plastic (control). Data are expressed as means ± SEM (n = 3). * p < 0.05; ** p < 0.01; **** p < 0.001 Mann–Whitney U test.

Article Snippet: For immunocytochemical analysis of cell proliferation, LMs were seeded at a density of 10,000 cells/well into 4 well-chamber slides (LabTek; Nunc, Wiesbaden, Germany), cultured for 72 h, stained with anti-Ki-67 antibody (Abcam; Cambridge, UK), and counted using Cell^F image analysis software (Olympus).

Techniques: In Vitro, Functional Assay, Blocking Assay, Migration, BrdU Incorporation Assay, Expressing, Incubation, Immunocytochemistry, Cell Culture, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Laminin-511-E8 promotes efficient in vitro expansion of human limbal melanocytes

doi: 10.1038/s41598-020-68120-0

Figure Lengend Snippet: List of antibodies used.

Article Snippet: For immunocytochemical analysis of cell proliferation, LMs were seeded at a density of 10,000 cells/well into 4 well-chamber slides (LabTek; Nunc, Wiesbaden, Germany), cultured for 72 h, stained with anti-Ki-67 antibody (Abcam; Cambridge, UK), and counted using Cell^F image analysis software (Olympus).

Techniques: Concentration Assay, Flow Cytometry, Blocking Assay, Immunohistochemistry, Immunocytochemistry