Journal: Retrovirology

Article Title: Differential resistance to cell entry by porcine endogenous retrovirus subgroup A in rodent species

doi: 10.1186/1742-4690-4-93

Figure Lengend Snippet: HuPAR topology . A . HuPAR-2 topology model derived by hydrophobicity algorithms and the experiments described in panel B-D is depicted. B-C . HuPAR-2 bearing an N- or C-terminal HA-tag was transiently transfected into 293T cells. After 48 hours cells were treated with saponin (intracellular staining) or without (surface staining). Following immunostaining using an anti-HA antibody and a FITC-conjugated secondary antibody, the samples were visualised either by confocal microscopy (B) or processed by flow cytometry (C). Immunostaining of the cells with anti-human CD71 was used as cell surface protein control. The cells nuclei were counter stained with propidium iodide. D . Cell lysates from 293T transiently transfected with an empty pcDNA3 (-), HuPAR-2 (wild type), HA-tagged HuPAR-2 wild type (C-HA wild type) or glycosylation mutant (C-HA N178A) were either treated (+) or untreated (-) with an enzyme removing N-linked oligosaccharide chains (PNGase F) and analysed by western blotting.

Article Snippet: The cells were washed twice in PBS, resuspended in PBS-2% FBS containing 1:100 dilution of mouse monoclonal antibody HA.11 (Covance) or 1 μg of mouse monoclonal anti-human CD71 antibody (Santa Cruz) and incubated for 1 hour at 4°C.

Techniques: Derivative Assay, Transfection, Staining, Immunostaining, Confocal Microscopy, Flow Cytometry, Control, Glycoproteomics, Mutagenesis, Western Blot

Journal: Cell Reports Methods

Article Title: Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells

doi: 10.1016/j.crmeth.2023.100460

Figure Lengend Snippet: HPCs generated in iSTC-Hp-spheroids can undergo maturation to erythroid cells, macrophages, or T lymphocytes using established cell maturation protocols (A) Schematic outline illustrating the use of Mart1-iPSC cells for iSTC-Hp-spheroid formation and subsequent erythroid cell, macrophage, and T lymphocyte differentiation. (B) Frequencies of CD34 + CD43 + and CD34 + CD45 + cells in Mart1-iPSC-derived iSTC-Hp-spheroids on day 13. Values represent mean ± SD from 3 independent experiments. (C–E) Mart1-iPSC-derived HPCs cultured in the erythroid differentiation condition for 15 days were harvested to characterize erythroid cell properties. The color of cell pellets changed to red (C), and typical erythroid lineage markers, such as CD71 and GPA, could be detected by flow cytometry analysis (D). Globin expression patterns were determined by relative RNA expression of α- and ζ-globin (α-globin series) and ε-, γ-, and β-globin (β-globin series) (E). Values represent mean ± SD from 3 independent experiments. (F–I) Macrophage differentiation from Mart1-iPSC-derived CD34 + cells. (F) Macrophage surface markers were analyzed by flow cytometry (gray; isotype control). (G) Giemsa stain of macrophage cytospin. Scale bar: 50 μm. (H) Phagocytosis assay of fluorescein isothiocyanate (FITC)-labeled zymosan A particles (gray; isotype control). (I) DHR assay of reactive oxygen species production by Mart1-iPSC-derived macrophages in response to stimulation with PMA (bottom panel); unstimulated macrophage control is also shown (top panel). (J–L) T lymphocytes generated from Mart1-iPSC-derived HPCs. (J) After 22 days of T cell differentiation, CD4 + CD8 + double-positive T cells could be detected by flow cytometry analysis, most of which were CD3 + Mart1-tetramer + cells. (K) Mart1-iPSC-derived CD4 + CD8 + double-positive T cells could be induced to CD8αβ single-positive T cells by stimulation with Mart1 peptide-primed T2 cells. (L) Mart1-iPSC-derived CD8 + single-positive T cells released interferon γ (INFγ) and tumor necrosis factor α (TNF-α) in response to Mart1 peptide-pulsed T2 cells. T cell responses without T2+Mart1 peptide (no target) or without Mart1 peptide (T2+DMSO) are also shown. Values represent mean ± SD from 3 independent experiments, ∗∗∗∗p < 0.0001.

Article Snippet: APC Mouse Anti-Human CD71 (Clone L01.1) , BD Biosciences , Catalog No: 341028, RRID AB_400560.

Techniques: Generated, Derivative Assay, Cell Culture, Flow Cytometry, Expressing, RNA Expression, Giemsa Stain, Phagocytosis Assay, Labeling, Cell Differentiation

Journal: Cell Reports Methods

Article Title: Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells

doi: 10.1016/j.crmeth.2023.100460

Figure Lengend Snippet:

Article Snippet: APC Mouse Anti-Human CD71 (Clone L01.1) , BD Biosciences , Catalog No: 341028, RRID AB_400560.

Techniques: Recombinant, Giemsa Stain, Modification, Cell Differentiation, Software