Journal: Nature biotechnology

Article Title: The mouse lymph node as an ectopic transplantation site for multiple tissues

doi: 10.1038/nbt.2379

Figure Lengend Snippet: Direct injection of hepatocytes into a single lymph node (LN) of a C57BL/6 wild type mouse. ( a ) Jejunal LN (yellow dotted oval) not transplanted (top panel) and just after transplantation (bottom panel) with hepatocytes. Primary hepatocytes were mixed with 3% Evans blue dye and Matrigel before injection. ( b ) In vivo optical imaging of animals transplanted at different sites. Primary hepatocytes from luciferase transgenic mice were transplanted in a single jejunal or popliteal LN, in spleen (SP) or intraperitoneally (IP). Signals (blue to red) depict different concentrations of donor hepatocytes at day 1 (top panels) and 1 week (bottom panels) after transplant. ( c ) Distribution of donor hepatocytes in a single jejunal LN. Top left panel, whole mount imaging of a LN one week after injection of donor GFP + hepatocytes. Bright field was merged with fluorescence. Top middle to bottom right panels, immunofluorescent (IF) staining of frozen LN serial sections with mAbs (red) against ER-TR7 (reticular fibroblasts), LYVE-1 (lymphatic vessels), PNAd (high endothelial venules), B220 (B cells) and CD4/CD8 (CD4 T and CD8 T cells) with the presence of GFP + hepatocytes (green). ( d ) IF staining of donor GFP+ hepatocytes (green) in jejunal LN one week after transplantation. Serial sections were stained with mAbs (red) against E-Cadherin (ECad), C-C chemokine receptor type 7 (CCR7) and sphingosine-1-phosphate receptor 1 (S1PR1). Nativel iver sections were stained as control (right panels). In native liver, CCR7 (red) was co-stained with dipeptidyl peptidase-4 (DPPIV) (green). All sections were counterstained with Hoechst 33342 (blue). ( e ) Proliferation of engrafted hepatocytes in LN after partial hepatectomy (PHx). Paraffin sections of injected LN and corresponding native liver one and two weeks after transplantation. Sections were stained for GFP (one and two weeks after transplantation) and BrdU (two weeks after transplantation), and revealed by peroxidase (brown cytoplasmic and brown nuclei staining respectively). Sections were counterstained with hematoxylin. Yellow outlines mark small populations of GFP + hepatocytes. 2 weeks after transplantation, bar graphs show the number of GFP + hepatocytes and % of BrdU + hepatocytes observed per section after immunostaining from animals with or without PHx.* P<0.05, ** P<0.0001. Data (mean + sem) are representative of one experiment with three to five mice per group. The experiment was repeated two times. Scale bar: (a) 1mm, (c–e) 100μm.

Article Snippet: Antibodies specific for the following antigens were purchased for immunohistochemistry: ER-TR7, LYVE1, GFP, Glutamine Synthetase, CCR7, S1PR1 (EDG1), F4/80 (Abcam), PNAd, B220, CD4 CD8 and Gr-1 (BD Biosciences), BrdU (Santa Cruz biotechnology), DPPIV (AbD Serotec), E-Cadherin (Zymed), Keratin 5 (Covance), Keratin 8 (DSHB), and C-peptide and Glucagon (Cell Signaling Technologies).

Techniques: Injection, Transplantation Assay, In Vivo, Optical Imaging, Luciferase, Transgenic Assay, Imaging, Fluorescence, Staining, Control, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Cytoplasmic anchorage of L-selectin controls leukocyte capture and rolling by increasing the mechanical stability of the selectin tether

doi: 10.1083/jcb.200103042

Figure Lengend Snippet: Rolling mediated by L Δ cyto mutant on endothelial and neutrophil ligands is impaired. (A) Tethering and rolling mediated by full-length L-selectin and LΔcyto mutant at low physiological shear stress (1 dyn/cm 2 ) on substrates coated with different densities of purified GlyCAM-1 or PNAd. Frequency values of tethering and rolling are depicted. Mean velocities of all rolling cells are indicated above the bars. (*) Rolling velocities not determined. (B) Tethering and rolling mediated by L-selectin and LΔcyto mutant interacting at moderate physiological shear stress (1.75 dyn/cm 2 ) on substrates coated with different densities of GlyCAM-1, PNAd. (C) Tethering and rolling of neutrophils on a monolayer of substrate-adherent L-selectin and LΔcyto mutant-transfected cells at different shear stresses. Values are the mean ± range of two experiments. (D) Tethering of neutrophils to single substrate-adherent transfectants expressing either L-selectin or LΔcyto measured at 1 or 1.75 dyn/cm 2 . Number and duration of tethers formed during 10-s perfusion of neutrophils (10 6 /ml) over six representative substrate-bound transfected cells are shown. All tethers were blocked in the presence of EGTA or the soluble L-selectin ligand, fucoidin.

Article Snippet: PNAd, purified from human tonsil lysates by MECA-79 mAb affinity chromatography , was a gift from Dr. E.L. Berg (Protein Design Labs).

Techniques: Mutagenesis, Shear, Purification, Transfection, Expressing