Journal: Nature biotechnology

Article Title: Development and function of human innate immune cells in a humanized mouse model

doi: 10.1038/nbt.2858

Figure Lengend Snippet: a , Percentages of human myeloid cells (hCD33 + ) among human hematopoietic cells (hCD45 + ) in the blood of the indicated recipient mice, engrafted as newborns by intra-hepatic injection of fetal liver CD34 + cells after X-ray preconditioning. Each symbol represents an individual mouse and the red bars indicate mean values (n=20-113; statistical analysis is shown in ). b , Composition of human white blood cells in the same mice as in a (n=20-113 mice/group; n=8 human donors; error bars indicate SEM). c , Immunohistological staining of human myeloid cells (hCD68 + ) in non-lymphoid tissues of the indicated recipient mice. The black bar represents 20 μm, and the images shown are representative of at least three mice analyzed per group. d-e , Representative flow cytometry analysis ( d ) and frequencies ( e ) of human monocyte subsets, identified by expression of CD14 and CD16 among hCD45 + CD33 + cells in the blood of recipient mice (n=8-12 mice/group; error bars indicate SEM). Dot plots in d are gated on CD33 hi SSC lo cells to show the subset distribution among monocytic cells. f , Human monocytes in the blood of MISTRG recipients and human monocytes from a human donor were stained with the indicated antibodies. Staining with isotype control antibodies is shown in red and specific antibodies in blue.

Article Snippet: Human CD33 + cells were enriched by magnetic isolation (EasySep CD33 selection kit, StemCell Technologies).

Techniques: Injection, Staining, Flow Cytometry, Expressing, Control

Journal: Molecular Human Reproduction

Article Title: ADAM8 localizes to extravillous trophoblasts within the maternal–fetal interface and potentiates trophoblast cell line migration through a β1 integrin-mediated mechanism

doi: 10.1093/molehr/gay034

Figure Lengend Snippet: ADAM8 preferentially localizes to anchoring columns of placental villi. (A) Representative immunofluorescence, immunohistochemistry and RNAScope microscopy images of serially sectioned first trimester placental villi (6–11 weeks gestation; n = 5) and decidua (10–12 weeks gestation; n = 4) showing ADAM8 mRNA transcript localization (pink punctate signal) within trophoblast subtypes. Proximal and distal column, as well as interstitial EVT subtypes of trophoblasts are identified by immunostaining of cytokeratin (KRT7) and HLA-G. ‘PCT’ indicates proximal column trophoblast; ‘DCT’ indicates distal column trophoblast; ‘VT’ indicates villous trophoblast; ‘SynT’ indicates syncytiotrophoblast; ‘MC’ indicates placenta mesenchymal core; ‘iEVT’ indicates interstitial extravillous trophoblast; ‘GC’ indicates trophoblast giant cell. The perforated white box indicates enlarged inset image. Black arrowheads denote ADAM8 signal. Bars = 100 μm. (B) Fluorescence activated cell-sorting (FACS) plots demonstrate the trophoblast isolation strategy used to purify mesencymal core cells (MC), distal column trophoblasts (DCT) and villous/proximal column trophoblasts (VT). Live cells, depleted of CD31+ (endothelial) and CD45+ (immune) cells using immuno-magnetic beads, were positively gated by 7AAD exclusion. Cells were further segregated by excluding CD45+ immune cells and by cell surface labeling of HLA-G and CD49f. Cell subtype proportions are indicated within each gated population (percent of cells within FACS plot). (C) ADAM8 mRNA levels in FACS-purified MCs, VTs and DCTs. Trophoblast subtype purity was assessed by qPCR analysis targeting the pan-trophoblast marker KRT7, the EVT-marker HLA-G and the mesenchymal lineage marker VIM. GAPDH was used for normalization. Results are presented as mean ± SD in bar graphs from four distinct placental villi specimens (n = 4); results were analyzed by one-way ANOVA and Dunn’s multiple comparisons test. (D) Immunoblot showing protein levels of pro- and active-ADAM8, HLA-G, and EGFR in MC, VT and DCT isolated from placental villi using immuno-magnetic beads. Molecular weights (kDa) are shown to the left and β-actin indicates loading control.

Article Snippet: Contaminating immune cells were removed from the cell admixture by EasySep CD45 immuno-magnetic bead purification (StemCell Technologies) where remaining cells were plated onto plastic culture plates for 30 min to remove stromal cells.

Techniques: Immunofluorescence, Immunohistochemistry, RNAscope, Microscopy, Immunostaining, Fluorescence, FACS, Isolation, Magnetic Beads, Labeling, Purification, Marker, Western Blot, Control