Journal: BMC Genomics

Article Title: Comparison of the molecular and cellular phenotypes of common mouse syngeneic models with human tumors

doi: 10.1186/s12864-019-6344-3

Figure Lengend Snippet: Immune subsets in syngeneic models. a In silico immune cell deconvolution of syngeneic tumor samples. Syngeneic models exhibited various immune cell type infiltrations with major NK cell infiltration predicted in CT26 models. b Comparison of estimated total T-cell fraction of leukocyte in selected mouse syngeneic models and their corresponding human tumors. Human data were downloaded from Gentles et al. . Total T-cell fraction plotted here is the sum of all predicted T-cell subsets including CD4+, CD8+, Treg, and gamma-delta T-cells. c CD3 staining for T-cells, CD11b staining for myeloid cells, and F4/80 staining for macrophage

Article Snippet: Sections were incubated with primary antibodies, CD11b (AbCam, EPR1334, Citrate, 0.088 μg/ml), F4/80 (Spring Bioscience, M4152, Citrate 2.5 μg/ml), RA3-6B2, Borg, 2.5 μg/ml), CD3 (Spring Bioscience, SP162, Borg, 1.33 μg/ml), vimentin (Cell Marque, SP20, Citrate, 3 μg/ml), E-cadherin (AbCam, EP700Y, Citrate, 0.18 μg/ml), for 1 h, washed in TBS and incubated with SignalStain Boost IHC Detection Reagent (Cell Signaling Technologies, Beverly, MA, USA) for 30 min.

Techniques: In Silico, Comparison, Staining

Journal: Experimental Animals

Article Title: NOD- Rag2 null IL-2Rγ null Mice: An Alternative to NOG Mice for Generation of Humanized Mice

doi: 10.1538/expanim.63.321

Figure Lengend Snippet: Percentages of T-cell subsets in the spleen, thymus, and peripheral blood of HSC-transplanted mice

Article Snippet: To identify human cells in the spleen of transplanted mice, the spleens were removed from the mice after blood removal, and tissues were fixed with 10 nM formalin (10 nM Mildform; Wako, Tokyo, Japan), embedded in paraffin, and stained with hematoxylin and eosin (HE), mouse anti-human CD45 antigen monoclonal antibody (clone 2B11+PD7/26; Dako Cytomation, Glostrup, Denmark), rabbit anti-human CD3 monoclonal antibody (clone SP7; Nichirei, Tokyo, Japan), and mouse anti-human CD79a monoclonal antibody (clone JCB117; Nichirei).

Techniques: Injection

Journal: Molecular Therapy. Nucleic Acids

Article Title: IL-10 modified mRNA monotherapy prolongs survival after composite facial allografting through the induction of mixed chimerism

doi: 10.1016/j.omtn.2023.02.016

Figure Lengend Snippet: IL-10 modRNA transcript produces functional protein in vitro (A–D) IL-10 modRNA produced IL-10 protein in mouse embryonic fibroblasts (MEFs), C2C12 mouse muscle myoblasts, human embryonic kidney 293 (HEK293) cells, and Jurkat cells (human T cells) at different time points by IL-10 ELISA. MEF (2 × 10 4 ), C2C12 (1 × 10 5 ), and 293 cells (1 × 10 5 ) were transfected with 100 ng modRNA. Jurkat cells (5 × 10 5 ) were transfected with 200 ng modRNA. All transfection experiments were performed in 96-well flat plate using Lipofectamine 3000 reagent. (E) Jurkat cells expressed IL-10 protein when transfected with IL-10 modRNA. Jurkat cells (12 × 10 6 ) were transfected without or with 5,000 ng IL-10 modRNA in 6-well plate. Cells were harvested one day after transfection, and 20 μg protein extract was analyzed using western blot assay. β-Actin was used as loading control. (F and G) Jurkat cells treated with IL-10 modRNA secreted functional IL-10 protein, which inhibited IL-2 and IFN-γ production in vitro . Jurkat cells (3 × 10 5 cells per well) transfected with 200 ng IL-10 modRNA were seeded in the top chambers of Transwell 96-well plates. Splenocytes (SPL; 3 × 10 5 cells per well) were seeded in bottom reservoir and stimulated with coated anti-CD3 (2 μg/mL) and soluble anti-CD28 (2 μg/mL). Supernatants in bottom reservoir were collected at different time points and analyzed using IL-2 and IFN-γ ELISA. Statistical data are represented as mean ± SD. The IL-10 modRNA group was compared with the buffer group at different time points (∗∗p < 0.005 and ∗∗∗p < 0.0005, Student’s t test).

Article Snippet: Primary antibodies were used against CD3 (SP7 clone; dilution 1:100; GeneTex) or FoxP3 (FJK-16 s clone; dilution 1:200; Thermo Fisher Scientific for 60 min.

Techniques: Functional Assay, In Vitro, Produced, Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: IL-10 modified mRNA monotherapy prolongs survival after composite facial allografting through the induction of mixed chimerism

doi: 10.1016/j.omtn.2023.02.016

Figure Lengend Snippet: Degree of lymphocytic infiltration into facial allograft (A) Macroscopic and histological changes (indicated by hematoxylin and eosin staining) in facial OMC allografts of IL-10 modRNA and buffer groups on PODs 10–14. Facial OMC allografts from allotransplanted mice receiving single 10 μg dose of IL-10 modRNA or saline buffer on POD 3 were analyzed. Infiltrating lymphocytes into skin and muscle layers of facial OMC allografts are shown by dense violet staining. Histological Banff classification of facial OMC allografts between buffer and IL-10 modRNA groups is as shown in the right of (A). Each group contains four mice. Scale bars, 50 μm. (B) Immunohistochemistry in facial OMC allografts of IL-10 modRNA and buffer groups on PODs 10–14. Infiltrating CD3 + T cells or FoxP3 + cells into skin and muscle layers of facial OMC allografts are shown by red staining. Because CD3 is expressed in the cell surface of T cells, the staining displays a circle red. FoxP3 is a critical transcription factor and marker for mouse CD4 + CD25 + natural Tregs, and the staining displays a dense red. Scale bars, 50 μm.

Article Snippet: Primary antibodies were used against CD3 (SP7 clone; dilution 1:100; GeneTex) or FoxP3 (FJK-16 s clone; dilution 1:200; Thermo Fisher Scientific for 60 min.

Techniques: Staining, Saline, Immunohistochemistry, Marker