Journal: Stem Cell Research & Therapy
Article Title: Human MuStem cells repress T-cell proliferation and cytotoxicity through both paracrine and contact-dependent pathways
doi: 10.1186/s13287-021-02681-3
Figure Lengend Snippet: Expression and secretion of immunoregulatory mediators in cultured human MuStem cells and bone marrow-derived mesenchymal stem cells. a Flow cytometry comparison of heme oxygenase-1 (HO-1) expression in hMuStem cells and BM-MSCs. b Fluorescent immunolabeling of inducible nitric oxide synthase (iNOS) in hMuStem cells and BM-MSCs. Lipopolysaccharide (LPS)-human monocyte-derived activated macrophages for 24 h and RAW 264.7 cell line were used as positive (C+) and negative (C−) controls, respectively. Nuclei were counterstained with DAPI (blue). Scale bars, 100 µm. c Representative RT-PCR profile of indoleamine 2,3-dioxygenase-1 (IDO-1) gene obtained for hMuStem cells and BM-MSCs. For each sample, the level of IDO-1 expression was quantified using the average mRNA level of IDO-1 obtained in unstimulated BM-MSCs as a reference. LPS-human monocyte-derived activated macrophages for 24 h and water were used as positive (C+) and negative (C−) controls, respectively. d Interleukin, growth factor, enzyme and carbohydrate-binding protein secretion profile of hMuStem cells and BM-MSCs. ELISA assays were performed using culture supernatant collected 24 h after medium change. Results are expressed as individual values and normalized as concentration relative to 1 million cultured cells (ng or pg/10 6 cells). Each experiment was performed on at least 5 and 4 independent batches of hMuStem cells and BM-MSCs, respectively. Stimulation corresponds to a 24-h treatment with 50 ng/mL of TNF-α and IFN-γ. * p < 0.05, ** p < 0.01, ** p < 0.001, **** p < 0.0001; Wilcoxon matched-pairs signed rank test (unstimulated vs. stimulated) or Mann–Whitney U test (hMuStem cells vs. BM-MSCs). US, unstimulated; S, TNF-α/IFN-γ-stimulated
Article Snippet: After incubation (1 h, RT) in blocking buffer (5% goat serum in PBS), cells were incubated overnight with iNOS Ab (1:100, sc-651 clone, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and counterstained (15 min, 37 °C) with DAPI fluorescent cell-permeable DNA probe (Life Technologies Ltd, Paisley, UK).
Techniques: Expressing, Cell Culture, Derivative Assay, Flow Cytometry, Comparison, Immunolabeling, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, MANN-WHITNEY
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