Journal: Science translational medicine

Article Title: Human limbal biopsy–derived stromal stem cells prevent corneal scarring

doi: 10.1126/scitranslmed.3009644

Figure Lengend Snippet: (A) Phase-contrast images of primary cells cultured from limbal biopsy tissue prepared by digestion with collagenase only. In initial plating (P0), red dashed lines mark islands of epithelial cells. Scale bars, 50 μm. (B) The length of time (in days) required to expand cells from P0 to P3 was compared for LBSCs prepared with dispase and collagenase (DSP + COL) or collagenase only (COL), expanding cells in either HS or FBS. Data are means ± SD (n = 4). P value determined by two-way analysis of variance (ANOVA). (C) Clonal growth of LBSCs in HS and FBS. (D) Percentage of clonogenic cells in P3 cultures. Data are means ± SD (n = 4). (E) Colony size was calculated with Fiji image analysis software. Data are means ± SD (n > 400). P values in (D) and (E) were determined by a two-sided t test. (F) Corneal fibroblasts in FBS did not exhibit clonal growth (n = 4).

Article Snippet: The remaining four segments were first incubated for 40 min in Dispase II neutral protease (Zen-Bio) at 37°C.

Techniques: Cell Culture, Software

Journal: Frontiers in Immunology

Article Title: Role of Src and Cortactin in Pemphigus Skin Blistering

doi: 10.3389/fimmu.2019.00626

Figure Lengend Snippet: Protective effect of Src inhibition against autoantibody-induced loss of cell cohesion is variable. (A) HaCaT cells were incubated with PV1-IgG or with control IgG (c-IgG) and subjected to dispase-based dissociation assays. Inhibition of Src by PP2 prevented fragmentation of cell monolayers after incubation with PV1 for 15 min and 60 min but not for 24 h ( n = 5; # p < 0.05; * p < 0.05 vs. c-IgG). (B) Western blot analysis revealed that Src was phosphorylated after 15, 30, and 120 min but not after 24 h of incubation with PV1 ( n = 3). (C–F) PV2- and PV3-IgG as well as AK23 were applied for several time points: 15 min (C) , 1 h (D) , 2 h (E) , and 24 h (F) , with keratinocytes being subsequently subjected to dissociation assays. Co-incubation with PP2 led to significantly reduced fragment numbers in PV3-IgG- and AK23- but not PV2-IgG-treated cells ( n > 7; # p < 0.05; * p < 0.05 vs. c-IgG). (G) Corresponding Western blot analysis for 2 h revealed that all autoantibody fractions were effective to activate Src ( n = 3).

Article Snippet: In brief, the skin was taken and incubated for 16 h in 2.4 U/ml dispase II in PBS supplemented with Gentamicin/AmphotericinB (CELLnTEC, Bern, Switzerland) at 4°C.

Techniques: Inhibition, Incubation, Control, Western Blot

Journal: Frontiers in Immunology

Article Title: Role of Src and Cortactin in Pemphigus Skin Blistering

doi: 10.3389/fimmu.2019.00626

Figure Lengend Snippet: Role of cortactin in Src-mediated modulation of AK23-induced loss of cell cohesion. (A) Cortactin is important for cell cohesion, indicated by enhanced fragmentation of cortactin-deficient (CTTN −/− ) cell monolayers in dispase-based dissociation assay ( n = 5; * p < 0.05 vs. control). (B) Confirmation of cortactin knockout by Western blot, α-Tubulin served as loading control ( n = 3). (C,D) CTTN −/− and wt mouse keratinocytes exposed to a dissociation assay following either short time (2 h) or long time (24 h) incubation with AK23 ( n > 7, # p < 0.05; * p < 0.05 vs. control). (E) Reconstitution of cell adhesion measured by dissociation assay after Ca 2+ switch in CTTN −/− and wt keratinocytes in absence or presence of Src inhibitor PP2 ( n > 7, # p < 0.05; * p < 0.05 vs. control).

Article Snippet: In brief, the skin was taken and incubated for 16 h in 2.4 U/ml dispase II in PBS supplemented with Gentamicin/AmphotericinB (CELLnTEC, Bern, Switzerland) at 4°C.

Techniques: Control, Knock-Out, Western Blot, Incubation