Journal: Life Science Alliance

Article Title: Fibro-adipogenic progenitors of dystrophic mice are insensitive to NOTCH regulation of adipogenesis

doi: 10.26508/lsa.201900437

Figure Lengend Snippet: (A) Schematic representation of cell isolation strategy. (B) Flow cytometry analysis of FAPs plated in growth medium (DMEM + 20% FBS) for 4 d and stained with antibodies raised against CD140a and α7-integrin. (C) CD31 − /CD45 − /a7-integrin − /SCA1 + cells were cultivated in growth and adipogenic media for 8 d and stained with antibodies against the MYHC and with DAPI. Scale bar: 100 μm. (D) Differentiation potential of CD31 − /CD45 − /a7-integrin − /SCA1 cells. Cells were cultivated for 8 d to differentiate spontaneously in growth medium or were induced to differentiate into adipocytes (adipogenic medium) or into fibroblasts (growth medium supplemented with 10 ng/ml TGFb). Adipocytes were identified with ORO stain, and fibroblasts were stained with fluorescent antibodies against smooth muscle actin (SMA). Nuclei were counterstained with DAPI. Scale bar: 100 μm. (E) Percentage of adipocytes and SMA covered area are presented on graphs. Quantification of the immunofluorescence images are presented as average ± SEM of three biological replicas (n = 3). Statistical significance was evaluated by the ANOVA test (* P < 0.05).

Article Snippet: For magnetic beads separation, the microbead-conjugated antibodies against CD45 (Cat. No. 130-052-301; Miltenyi Biotech), CD31 (Cat. No. 130-097-418; Miltenyi Biotech), α7-integrin (Cat. No. 130-104-261; Miltenyi Biotech), and SCA1 (Cat. No. 130-106-641; Miltenyi Biotech) were used.

Techniques: Cell Isolation, Flow Cytometry, Staining, Immunofluorescence

Journal: Life Science Alliance

Article Title: Fibro-adipogenic progenitors of dystrophic mice are insensitive to NOTCH regulation of adipogenesis

doi: 10.26508/lsa.201900437

Figure Lengend Snippet: FAPs were purified using MACS microbead technology from hind limb muscles of 6-wk-old wt , mdx , and ctx (3 dpostinjury) mice. The different cell samples were bar coded by labeling with different proportions of three palladium isotopes. The bar coded cell samples were pooled and incubated with 12 metal-labeled antibodies (SCA1, CD34, CD146, CD140b, CD31, CXCR4, CAS3, Vim, pSTAT1, TNFa, pCREB and pSTAT3) and analyzed by mass cytometry in a Cytof2 instrument. (A) Schematic representation of the experimental design. (B) Intensity distribution of the signals of four different antigens (SCA1, CD34, CD146 and CD140b). (C) Contour maps of the scatter plots showing the correlations between SCA1 and CD34 expression in the three biological repeats for the three conditions. Colors represent cell densities:red = high, blue = low. The two different identified subpopulations are outlined by green (SCA1 H CD34 H ) or yellow (SCA1 L CD34 L ) ovals. (D) Contour representations of the viSNE maps in the nine different cell preparations. Regions of interest identifying cell subpopulations that characterize the different biological conditions are circled (A to E in the top left panel).

Article Snippet: For magnetic beads separation, the microbead-conjugated antibodies against CD45 (Cat. No. 130-052-301; Miltenyi Biotech), CD31 (Cat. No. 130-097-418; Miltenyi Biotech), α7-integrin (Cat. No. 130-104-261; Miltenyi Biotech), and SCA1 (Cat. No. 130-106-641; Miltenyi Biotech) were used.

Techniques: Purification, Muscles, Labeling, Incubation, Mass Cytometry, Expressing

Journal: Life Science Alliance

Article Title: Fibro-adipogenic progenitors of dystrophic mice are insensitive to NOTCH regulation of adipogenesis

doi: 10.26508/lsa.201900437

Figure Lengend Snippet: The single-cell multiparametric data obtained in the mass cytometry experiment described in was projected onto a two-dimensional space by applying the viSNE algorithm . (A) List of metal-tagged antibodies used in the mass cytometry experiment. (B) Dot representations of the viSNE maps, where each dot represents a cell. In the different maps, dots are colored according to the intensity of expression of the 12 antigen readouts (SCA1, CD34, CD146, CD140b, CD31, CXCR4, CAS3, Vim, pSTAT1, TNF-α, pCREB and pSTAT3). (C) Bar plots representing the relative abundance of the two subpopulations expressing different levels of SCA1 and CD34.

Article Snippet: For magnetic beads separation, the microbead-conjugated antibodies against CD45 (Cat. No. 130-052-301; Miltenyi Biotech), CD31 (Cat. No. 130-097-418; Miltenyi Biotech), α7-integrin (Cat. No. 130-104-261; Miltenyi Biotech), and SCA1 (Cat. No. 130-106-641; Miltenyi Biotech) were used.

Techniques: Mass Cytometry, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TCR Retrogenic Mice as a Model to Map Self-Tolerance Mechanisms to the Cancer Mucosa Antigen Guanylyl Cyclase C (GUCY2C)

doi: 10.4049/jimmunol.1801206

Figure Lengend Snippet: A–C) Bone marrow was isolated from donor Rag1−/− mice and HSCs were purified by negative selection of lineage-positive cells. Purified HSCs were left untransduced or transduced 1–3 times with DO11 or GUCY2C TCRs. A) HSC expansion ex vivo. B) Transduced HSCs remained lineage negative (mature lineage markers B220, Ter119, CD4, and Gr-1 shown here) following retroviral transduction and expansion, comparable to HSCs stained directly ex vivo. C) HSC transduction efficiency following 1, 2, or 3 applications of TCR retrovirus (1x, 2x or 3x). Values indicate percentage of GFP+ cells. Data are representative of 3 experiments.

Article Snippet: Briefly, Rag1 − /− mice were injected i.p. with 150 mg/kg of 5-fluorouracil (Millipore Sigma); bone marrow was harvested 72 h later and murine HSCs were purified using mouse HSC isolation kit and EasySep magnet (STEMCELL, Vancouver, BC, Canada).

Techniques: Isolation, Purification, Selection, Ex Vivo, Transduction, Staining