celltrace violet proliferation dye ctv  (Thermo Fisher)

Journal: Cancer letters

Article Title: PKC agonism restricts innate immune suppression, promotes antigen cross-presentation and synergizes with agonistic CD40 antibody therapy to activate CD8 + T cells in breast cancer

doi: 10.1016/j.canlet.2022.01.017

Figure Lengend Snippet: (A – D) BM cells collected from tumor-free C57Bl/6J mice were cultured in vitro in the presence of granulocyte-macrophage colony stimulating factor GM-CSF (20 ng/mL) and E0771 conditioned media (CM) (50% V/V) for 4 days in the presence of Veh or PEP to generate MDSCs. (A) Experimental outline and gating scheme. (B) Representative flow plot of CD11b + Gr-1 + cells in Veh- or PEP-treated BM cultures. (C) Frequencies and (D) absolute cell numbers of CD11b + Gr-1 + cells (n = 4 mice). Shown are Means ± SEM. Unpaired t-test (*P < 0.05, ***P < 0.005). (E) Ccr2 gene expression in M-MDSC isolated from the spleens (sM-MDSC) of E0771 tumor-bearing mice and treated ex vivo with PEP (20 nM) or Veh for 72h as analyzed by qRT-PCR (n = 4 mice). Shown are Means ± SEM. Unpaired t-test (****P < 0.001). (F–I) Splenic CD11b + Gr-1 + MDSCs (sMDSCs) were isolated from E0771 tumor-bearing mice and co-cultured with CellTrace Violet (CTV)-labeled CD8 + T cells isolated from the spleens of tumor-free mice at a 1:2 T cell/MDSC ratio. sMDSCs were pre-treated with PEP (20 nM) or Veh for 3h prior to the addition of T cells and Dynabeads (anti-CD28/CD3). MDSC suppressive properties were assessed by CD8 + T cell activation (IFNγ expression) and proliferation (CTV dilution). (F) Representative contour plots and (G) IFNγ expression in CD8 + T cells (n = 3 mice). One-way ANOVA with multiple comparisons with Tukey correction (***P < 0.0001). (H–I) sPMN-MDSCs and sM-MDSCs were isolated from the spleens of tumor-bearing mice as previously described. Percent of CD8 + T cell proliferation after coculture with Veh- or PEP (20 nM)-pre-treated (H) sPMN-MDSCs and (I) sM-MDSCs at the indicated T cell:MDSC ratios (n = 3 mice). Shown are Means ± SEM. (**P < 0.01, ***P < 0.005).

Article Snippet: Isolated CD8 + T cells were labeled with CellTrace Violet Cell Proliferation Dye (CTV, Thermo Fisher) and were added to MDSCs (5×10 4 CD8 + T cells/well).

Techniques: Cell Culture, In Vitro, Gene Expression, Isolation, Ex Vivo, Quantitative RT-PCR, Labeling, Activation Assay, Expressing