[PMC free article] [PubMed] [CrossRef] [Google Scholar] 10. to excised colorectal carcinoma samples for mechanical fragmentation, enzyme digestion, and Percoll density gradient centrifugation (GE Healthcare). The granulocyte portion was harvested, and CD117+ mast cells were positively selected using anti-CD117 or anti-FcR1 antibody-coated magnetic beads (Fig. 1A). In the anti-CD117 antibody-enriched cells, 97% of the cells offered a CD203c+ phenotype, and no CD163L1 or little expression of CD123 was observed (Fig. 1B). All cells showed a tryptase-positive reaction on intracellular staining, and the majority of purified cells expressed the high-affinity IgE receptor FcR1 and displayed binding with soluble IgE immunoglobulin (Fig. 1B). Tryptase is one of the granule components of mast cells and could be observed by confocal microscopy of intracellular staining (Fig. 1C), and ongoing degranulation of cells was also observed after toluidine blue staining (Fig. 1D). Under transmission electron microscopy, purified cells exhibited a characteristic phenotype, with the monolobed nuclei and numerous thin, elongated folds round the cells (Fig. 1E) that are common of mast cells (31). Open in a separate windows FIG 1 Characteristics of intestinal mucosal mast cells. (A) Enrichment and purification of mucosal mast cells from human healthy colorectal tissues. (B) Phenotype of purified mast cells as analyzed by immunostaining with specific antibodies and circulation cytometry. (C) Intracellular immunostaining of tryptase (reddish) was confirmed by confocal microscopy; nuclei were stained with DAPI. DIC, differential interference contrast. (D) Positive staining of mast cells by toluidine blue. (E) Visualization of mast cells by transmission electron microscopy. Human mucosal mast cells express HIV-1 attachment factors for viral capture. To investigate the conversation of mast cells with HIV-1, we first explored the binding of viruses to cells. Freshly isolated mast cells were pulsed with HIV-1-gag-GFP/JRFL VLPs, and VLPs/Env, which do not incorporate HIV-1 envelope proteins, were used to monitor nonspecific binding. Viral association was quantified by circulation cytometry to detect green fluorescent protein (GFP) levels. At 4C, about 22.3% of mast cells were found to capture JRFL VLPs, and no obvious binding was observed with VLPs/Env, indicating that the binding was envelope dependent and Oxymatrine (Matrine N-oxide) that the cell-associated HIV-1 particles could be removed by trypsin treatment (Fig. 2A). Confocal microscopy was also used to visualize and confirm viral surface binding (Fig. 2B), and replication-competent HIV-1 AD8 was utilized to imagine the binding of pathogen to mast cells by TEM (Fig. 2C). To verify that HIV-1 binding can be envelope dependent, the binding was examined by us of recombinant HIV-1 gp120 glycoprotein to mast cells. As demonstrated in Fig. 2D, HIV-1 JRFL-derived gp120 glycoproteins had been discovered to bind to mast cells. Open up in another home window FIG 2 Intestinal mucosal mast cell-mediated HIV-1 catch. (A) Recognition of HIV-1 VLP binding on mast cells by movement cytometry. VLPs including Gag-GFP had been pulsed with mast cells at 4C, and VLPs/Env had been utilized as the control to monitor non-specific binding. Trypsin treatment was Oxymatrine (Matrine N-oxide) utilized to eliminate surface-bound infections. (B) HIV-1 VLP association with cells was noticed by confocal microscopy. (C) Binding of replication-competent HIV-1 Advertisement8 on mast cells as visualized by TEM. Arrows reveal infections. (D) Binding of gp120 on mast cells. Purified mast cells had been cultured with recombinant gp120 glycoproteins for 1 h at 4C and set for immunostaining and recognized by movement cytometry. (E) Manifestation of HIV-1 connection factors as recognized by immunostaining with particular antibodies and movement cytometry. (F) Colocalization of HIV VLPs with DC-SIGN, HSPG, or 47 integrin. Purified mast cells had been incubated with HIV-Gag-GFP/JRFL VLPs (40 ng p24gag) for Oxymatrine (Matrine N-oxide) 1 h at 4C and seeded onto poly-l-lysine-coated microscope slides. Cells had been immunostained and set with particular antibodies against human being DC-SIGN, HSPG, 4, or 7, accompanied by secondary Alexa.