prolong gold antifade mounting media  (Thermo Fisher)

Journal: Cells

Article Title: Regulatory T Cell Mimicry by a Subset of Mesenchymal GBM Stem Cells Suppresses CD4 and CD8 Cells.

doi: 10.3390/cells14080592

Figure Lengend Snippet: Figure 4. Pharmacological TGFBR2 inhibition attenuates immunosuppressive phenotype of GSCs. (A) Representative immunofluorescence images (left) and quantification (right) showing TGFBR2 expression in M1123 cells 24 h after treatment with ITD-1 (20 µM) or vehicle control (DMSO). Scale bar = 50 µm. (B) qRT-PCR analysis showing expression of Treg effector genes 72 h following treatment with ITD-1 (20 µM) or vehicle control (DMSO). (C) CD4+ and CD8+ T cell viability was measured 48 h after culture in media conditioned with GSCs treated with ITD-1 or DMSO. CD4+ and CD8+ T cells cultured in unconditioned (UC) media were used as control. (D) Tumor cell death was measured via caspase 3/7 assay 48 h after co-culturing with CD4+ or CD8+ T cells cultured in media conditioned with GSCs pre-treated with ITD-1 or DMSO as described in Materials and Methods. Tumor cells cultured in absence of CD4+ or CD8+ T cells were used as baseline control. (E) Representative immunofluorescence images (left) and quantification (right) of PD1 expression in CD8+ T cells 48 h after culturing in media conditioned with GSCs treated with ITD-1 or DMSO. Scale bar = 50 µm. PD1 fluorescence intensity was measured using ImageJ v1.51 ‘Analyze Particles’ feature and normalized to total DAPI intensity in multiple fields of view (n = 20 per condition). Statistical significance was calculated using Mann–Whitney U-test for panel A, Student’s t-test for panel (B), one-way ANOVA with Tukey’s post hoc test for panels (C–E). Data are shown as mean ± SD for all bar graphs. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Data shown in panels (B–E) were gathered in triplicate and are representative of 2 independent experiments.

Article Snippet: The next day, cells were washed with PBS and coverslips were mounted with Prolong Gold Antifade plus DAPI mounting media (Cell Signaling, Danvers, MA, USA).

Techniques: Inhibition, Immunofluorescence, Expressing, Control, Quantitative RT-PCR, Cell Culture, Fluorescence, MANN-WHITNEY