Fig. 6
Establishment and confirmation of vascularized TSG6 knockdown spheroids of canine MGT cells with canine ECs. (A) Method of forming vascularized TSG6 knockdown spheroids comprising of canine ECs and MGT cells. Cells were seeded onto an ultra-low adhesion plate with a 1:3 ratio. (B) Characterization of canine ECs by flow cytometry. Canine ECs were positive for CD31. (C) Confocal imaging of vascularized TSG6 knockdown spheroids. The spheroids were stained with APC dye-conjugated IgG1 mouse monoclonal anti-pimonidazole antibody (red), anti-CD31 rabbit monoclonal antibody (green), and Hoechst 33,342 (blue) (scale bar = 100 μm). (D) Immunohistology images of vascularized TSG6 knockdown spheroids. The spheroids were stained with anti-CD31 rabbit monoclonal antibody (brown) (scale bar = 20 μm). (E) Relative CD31-positive area in vascularized TSG6 knockdown spheroids. The results are presented as the mean ± SD and were of three obtained independent experiments. TSG6, TNF-stimulated gene 6 protein; MGT, mammary gland tumor; EC, endothelial cell; APC, Allophycocyanin; IgG1, Immunoglobulin G1; ctrl, control. *p < 0.05, **p < 0.01, as determined by one-way ANOVA.
