Journal: Cancers

Article Title: Wilms’ Tumor Primary Cells Display Potent Immunoregulatory Properties on NK Cells and Macrophages

doi: 10.3390/cancers13020224

Figure Lengend Snippet: Evaluation of IL2 activated NK cell cytotoxic activity before and after co-culture with WT primary cultures ( A ) The cytotoxicity assay of NK cells from healthy donors cultured alone or with WT cell lines in the presence of IL2 on the left and alone or with K562 cells in presence of IL2 on the right. After six days, NK cells were incubated with K562 target cells for 4 h at the indicated E:T ratio. Data shown here are the average of four independent experiments for each WT cell line ± SEM. Statistical significance was determined by Student’s t -test (* p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001). ( B ) mRNA fold change of activating receptors (NKp46, NKp44, NKp30, and DNAM-1) and effector molecules (IFNγ, TNFα, Granzyme B, and Perforine1). The results are the means ±SEM of three different experiments with different NK donors. ( C ) NK cell surface expression of CD107a and intracellular expression of IFN-γ after six days of culture alone or with WT cell lines under direct contact. The results are the mean ± SEM of three different experiments for each WT cell line. p -values were calculated using one-way ANOVA with Fisher’s LSD multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001). ( D ) Surface expression of DNAM-1, NKp30 and NKp46 were measured by flow cytometry in NK cells on day six of culture with IL-2 alone or in presence of WT cells. Bar graphs show the mean fluorescence intensity (MFI) ratio between stained and unstained cells. The results are the means ± SEM of three different experiments for each WT cell line. p -values were calculated using one-way ANOVA with Fisher’s LSD multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Staining was performed on 5-μm TMA sections (KD2084 Renal disease spectrum tissue array, US Biomax, Inc.) using a mouse monoclonal anti-NKp46 primary antibody (anti-NKp46/NCR1 195,314 R&D Systems for immunohistochemistry assay) and automated BenchMark ULTRA slide stainer, according to the manufacturer’s protocol.

Techniques: Activity Assay, Co-Culture Assay, Cytotoxicity Assay, Cell Culture, Incubation, Expressing, Flow Cytometry, Fluorescence, Staining

Journal: Respiratory Research

Article Title: Following damage, the majority of bone marrow-derived airway cells express an epithelial marker

doi: 10.1186/1465-9921-7-145

Figure Lengend Snippet: Widefield visualisation of Y positive donor-derived cells and expression of CD45 and cytokeratin fall into four classes . CD45 positive stained cells (CY3-red) were identified on tracheal sections, photographed, and their positions recorded. The CD45 signal was lost in subsequent manipulations. Sections were then subjected to Y chromosome FISH (Tx-Red) and immunohistochemistry for cytokeratin (FITC-green). Donor-derived (Y FISH positive) cells fell into four classes (Table 2). Figure 1A shows a CD45 positive (i) and cytokeratin positive (ii) coincident cell arrowed; CD45 positive (iii) and cytokeratin negative (iv) coincident cell arrowed; CD45 negative (not shown) and cytokeratin positive (v,vi,vii,viii); CD45 negative (not shown) and cytokeratin negative (ix, x) arrowed cells. ix also contains a cytokeratin positive cell marked with an arrowhead. 1B top panel shows two CD45 positive (i) and cytokeratin positive (ii) cells and the brightfield staining of this tracheal section (iii) with cells marked with arrows; Figure 1B bottom panel shows a CD45 positive (iv), cytokeratin negative (v) cell and photomicrograph (vi) shows brightfield staining of the same region with the cell marked with arrowhead. shows cytokeratin staining alone of tracheal sections and also cytokeratin staining after Y chromosome FISH illustrating that the distribution of cytokeratin staining is the same. Scale bar 5 uM.

Article Snippet: Spleen was used as a positive control for CD45 as detailed on IHC World online information center for immunohistochemistry [ ].

Techniques: Derivative Assay, Expressing, Staining, Immunohistochemistry

Journal: Respiratory Research

Article Title: Following damage, the majority of bone marrow-derived airway cells express an epithelial marker

doi: 10.1186/1465-9921-7-145

Figure Lengend Snippet: Deconvolved visualisation of cells with Y chromosome FISH and cytokeratin immunohistochemistry . Examples of Y chromosome positive cells by Y FISH (Tx-Red) and cytokeratin immunohistochemistry (FITC-green). Arrows mark cells positive for Y chromosome and either positive for cytokeratin (A,B,C,D) or negative (E and F). Videos of positive and negative cells can be found in additional files (cytokeratin positive movie) and (cytokeratin negative movie).

Article Snippet: Spleen was used as a positive control for CD45 as detailed on IHC World online information center for immunohistochemistry [ ].

Techniques: Immunohistochemistry

Journal: Respiratory Research

Article Title: Following damage, the majority of bone marrow-derived airway cells express an epithelial marker

doi: 10.1186/1465-9921-7-145

Figure Lengend Snippet: Co-localisation of Y FISH and cytokeratin immunosignal in single cells . Female animals engrafted with male bone marrow were sacrificed after three months and cells dissociated from the tracheas were cytospun onto slides and subjected to Y FISH (red) and cytokeratin immunocytochemistry (green). Y positive (donor derived) cells are arrowed and both Y chromosome positive and cytokeratin negative (panels A,G and I) and Y positive, cytokeratin positive cells (panel B,C and E) were observed. Non-arrowed cells in panels A and B are cytokeratin positive but not Y FISH positive and presumably host female derived. Panels C and G show donor cells (arrow) stained for presence of Y chromosome and then the slides were counterstained with H and E (panel D and H respectively). Panels E and I show the donor Y FISH positive cells (arrowed) with panels F and J showing the same cells stained with H and E. The donor cell (arrowed) in panels I and J is cytokeratin negative and has macrophage morphology.

Article Snippet: Spleen was used as a positive control for CD45 as detailed on IHC World online information center for immunohistochemistry [ ].

Techniques: Immunocytochemistry, Derivative Assay, Staining