cd34 specific ab  (Bioss)

Journal: Science Advances

Article Title: Identification of a humanized mouse model for functional testing of immune-mediated biomaterial foreign body response

doi: 10.1126/sciadv.ade9488

Figure Lengend Snippet: 500-μm-diameter SLG20 alginate spheres (0.5-ml volume) were implanted into the IP space of C57BL/6 and various humanized mouse models for 28 days and then analyzed for the degree of fibrosis. ( A ) Graphic showing steps required to achieve fibrotic FBR in the human HSC–engrafted NSG-SGM3 BLT model. BM, bone marrow; IV, intravenous. ( B ) Bright-field images of retrieved spheres reveal significant overgrowth in control C57BL/6 mice (left), with no fibrosis in NSG BLT mice, without or with xenogeneic neonatal pig cell clusters (NPCCs) (middle images). FBR was only observed after transgenic expression of three human cytokines [SCF, GM-CSF, and IL-3 (SGM3)] for innate immune cell maintenance. ( C ) Bright-field images (4× and 10×) of fibrosed spheres retrieved from engrafted NSG-SGM3 BLT mice 28 days after implantation. ( D ) Collagen levels on retrieved capsules by hydroxyproline assay from nonengrafted NSG-SGM3 BLT (white), wild-type C57BL/6 (gray), or engrafted (humanized) NSG-SGM3 BLT mice (red). ( E ) Phase contrast images of fibrosed and epididymal fat-embedded 5-mm PDMS discs and 500-μm PS spheres retrieved 28 days after IP implantations (alginate in fig. S1B). ( F ) Flow cytometry analysis reflected similar CD45 + engraftment without (blue) versus with (red) SGM3 incorporation in the blood, spleen, and bone marrow; however, a significant increase occurred in the IP space. PEC, peritoneal exudate cell. ( G ) With SGM3 adoption, CD45 + CD3 − CD20 − CD33 + innate myeloid lineages were significantly increased in all compartments. For statistical analysis, one-way analysis of variance (ANOVA) with Bonferroni multiple comparison correction was used. * P < 0.05 and *** P < 0.0001; ns, not significant. n = 5 (biologic replicates) per group. Experiments were repeated three times.

Article Snippet: Implanted mice were subsequently injected intravenously with 1 × 10 5 CD34 + autologous fetal liver-derived HSCs (determined by flow cytometry with a CD34-specific Ab; clone 581, BD Biosciences) between 4 to 6 hours after irradiation, as described previously ( ).

Techniques: Control, Transgenic Assay, Expressing, Capsules, Hydroxyproline Assay, Flow Cytometry, Comparison

Journal: Science Advances

Article Title: Identification of a humanized mouse model for functional testing of immune-mediated biomaterial foreign body response

doi: 10.1126/sciadv.ade9488

Figure Lengend Snippet: ( A ) As compared to nonfibrosed (nonengrafted) and fibrosed (engrafted) controls, clodrosome depletion of human macrophages resulted in complete loss of fibrosis on 500-μm-diameter SLG20 alginate spheres following 2-week IP implantations in engrafted NSG-SGM3 BLT mice. ( B ) Flow cytometry analysis of human immune cell numbers (#) dissociated from capsule spheres (Caps) or PECs retrieved 2 or 4 weeks after IP implantation (green versus blue, respectively) showing significant increases of human (h)CD45 + CD3 − CD20 − CD33 + myeloid cells, which were eliminated following clodrosome macrophage (Mϕ) depletion throughout 2-week implantations (red). By comparison, human neutrophils (CD45 + CD66b + ) did not significantly change. ( C ) As compared to very high absolute human immune cell numbers [in (B)], effective mouse immune cell elimination by irradiation was confirmed by low counts of residual mouse (m) macrophages (F4/80 + CD11b + ) and neutrophils (Ly6g + CD11b + ), with no significant response by either population to material implantation or macrophage depletion. ( D ) NanoString analysis of cell and cytokine markers on alginate spheres at 1, 5, and 14 days after implantation in engrafted NSG-SGM3 BLT mice versus non-engrafted and engrafted but macrophage-depleted controls. Similar to wild-type FBR, macrophage and B cell markers were some of the largest dynamic responders. White, within background of the assay. ( E ) No human collagen was observed; instead, delayed mouse αSMactin fibroblast and collagen (Col1a1) expression indicated mouse myofibroblast engagement. For all, n = 5 (biologic replicates) per treatment. Flow cytometry was performed twice, and NanoString was performed once. For flow comparisons, one-way ANOVA (*** P < 0.0001 versus clodrosome-depleted controls) was used. For NanoString, log 2 scale was used; for statistical analysis, see Materials and Methods.

Article Snippet: Implanted mice were subsequently injected intravenously with 1 × 10 5 CD34 + autologous fetal liver-derived HSCs (determined by flow cytometry with a CD34-specific Ab; clone 581, BD Biosciences) between 4 to 6 hours after irradiation, as described previously ( ).

Techniques: Flow Cytometry, Comparison, Irradiation, Expressing