Autonomous regulation of IgE-mediated mast cell degranulation and immediate hypersensitivity reaction by an inhibitory receptor CD300a
Authors: Yaqiu Wang, Chigusa Nakahashi-Oda, Yoshimichi Okayama, Akira Shibuya
Journal: Journal of Allergy and Clinical Immunology (2019)

Although phosphatidylserine (PS) confined to the inner leaflet of plasma membrane is exposed on the cell surface when cells undergo apoptosis, viable cells, including mast cells (MCs), also externalize PS in certain cellular states.E1Lentz B. Exposure of platelet membrane phosphatidylserine regulates blood coagulation.Prog Lipid Res. 2003; 42: 423-438Crossref PubMed Scopus (260) Google Scholar, E2Segawa K. Suzuki J. Nagata S. Constitutive exposure of phosphatidylserine on viable cells.Proc Natl Acad Sci USA. 2011; 108: 19246-19251Crossref PubMed Scopus (131) Google Scholar, E3Elliott J.I. Sardini A. Cooper J.C. Alexander D.R. Davanture S. Chimini G. et al.Phosphatidylserine exposure in B lymphocytes: a role for lipid packing.Blood. 2006; 108: 1611-1617Crossref PubMed Scopus (52) Google Scholar, 1Elliott J. Surprenant A. Marelli-Berg F. Cooper J. Cassady-Cain R. Wooding C. et al.Membrane phosphatidylserine distribution as a non-apoptotic signalling mechanism in lymphocytes.Nat Cell Biol. 2005; 7: 808-816Crossref PubMed Scopus (187) Google Scholar, 2Martin S. Pombo I. Poncet P. David B. Arock M. Blank U. Immunologic stimulation of mast cells leads to the reversible exposure of phosphatidylserine in the absence of apoptosis.Int Arch Allergy Immunol. 2000; 123: 249-258Crossref PubMed Scopus (83) Google Scholar However, the pathophysiological significance of PS exposure on viable cells remains elusive. To address the role of PS externalization on the cell surface of viable MCs, we monitored PS surface exposure on bone marrow–derived cultured MCs (BMMCs) by confocal microscopy after stimulation with trinitrophenyl (TNP)-specific IgE and TNP-ovalbumin (OVA) in the presence of PSVue 643, a fluorescent dye with rapid binding capacity for PS. The dye began to accumulate on the cell surface of live BMMCs within 600 seconds after FcεRI stimulation, whereas the nonstimulated BMMCs remained negative for the staining (Fig 1, A; see Video E1 in this article's Online Repository at www.jacionline.org), indicating that PS is externalized within 10 minutes after activation. MCs abundantly express CD300a, an inhibitory immunoreceptor for PS.3Yotsumoto K. Okoshi Y. Shibuya K. Yamazaki S. Tahara-Hanaoka S. Honda S. et al.Paired activating and inhibitory immunoglobulin-like receptors, MAIR-I and MAIR-II, regulate mast cell and macrophage activation.J Exp Med. 2003; 198: 223-233Crossref PubMed Scopus (77) Google Scholar, 4Nakahashi-Oda C. Tahara-Hanaoka S. Honda S. Shibuya K. Shibuya A. Identification of phosphatidylserine as a ligand for the CD300a immunoreceptor.Biochem Biophys Res Commun. 2012; 417: 646-650Crossref PubMed Scopus (42) Google Scholar, 5Nakahashi-Oda C. Tahara-Hanaoka S. Shoji M. Okoshi Y. Nakano-Yokomizo T. Ohkohchi N. et al.Apoptotic cells suppress mast cell inflammatory responses via the CD300a immunoreceptor.J Exp Med. 2012; 209: 1493-1503Crossref PubMed Scopus (64) Google Scholar By imaging flow cytometry analyses, we found that both PS and CD300a showed a polarization and colocalization on the cell surface of mouse BMMCs (Annexin V–stained) and cultured human synovial MCs (hMCs) 15 minutes after FcεRI stimulation (Fig 1, B; see Fig E1, A and B, in this article's Online Repository at www.jacionline.org). Fluorescence resonance energy transfer (FRET) analyses using 1-oleoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoserine (NBD-PS) as a donor and a non-neutralizing anti-CD300a antibody (TX10) conjugated with Alexa546 as an acceptor demonstrated increased FRET efficiency calculated by sensitized emission between CD300a and PS during degranulation (Fig 1, C). Treatment with a neutralizing anti-CD300a antibody (EX42) that interferes with the binding of CD300a with PS dramatically decreased the FRET+ cell number during degranulation (see Fig E1, C and D), demonstrating the direct cis-interaction between CD300a and PS in the colocalized region of degranulating MCs. PS also showed a colocalization with CD107a on the cell surface of degranulating (ie, CD107a+ Annexin V+) BMMCs (see Fig E1, E), suggesting that PS exposure on the cell surface of MCs is associated with degranulation. Indeed, treatment of BMMCs with the MC- degranulating stimuli ATP and ionomycin induced the exposure of PS as well as CD107a on the cell surface. In contrast, neither CD107a nor PS was observed on the cell surface of BMMCs after stimulation with either MC activator LPS or IL-336Joulia R. L'Faqihi F.-E. Valitutti S. Espinosa E. IL-33 fine tunes mast cell degranulation and chemokine production at the single-cell level.J Allergy Clin Immunol. 2017; 140: 497-509.e10Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar (see Fig E1, F). Unlike FcεRI stimulation, however, stimulation with neither ATP nor ionomycin induced the polarization of CD300a and PS on the cell surface of MCs (see Fig E1, G). Together, these data demonstrate that PS is promptly exposed together with CD107a during degranulation of MCs and colocalized with CD300a. We next examined the degranulation of wild-type (WT) and Cd300a−/− BMMCs following stimulation of FcεRI by addition of TNP-specific IgE and TNP-OVA. Flow cytometry analyses showed that the size of the CD107a+ population was significantly larger in Cd300a−/− BMMCs than in WT BMMCs from 8 minutes (480 seconds) to 30 minutes (1800 seconds) after stimulation (Fig 2, A and B). To confirm the cell-intrinsic effect of CD300a on degranulation, equal numbers of WT and Cd300a−/− BMMCs were mixed and stimulated with TNP-specific IgE and TNP-OVA. The CD107a+ population was again observed to be larger in Cd300a−/− BMMCs than in WT BMMCs (see Fig E2, A and B, in this article's Online Repository at www.jacionline.org). Moreover, Cd300a−/− BMMCs released a larger amount of β-hexosaminidase than did WT BMMCs when they were analyzed 30 minutes after stimulation (Fig 2, C). We found that the population of propidium iodide (PI)+ dead cells was comparable between the cultures of WT and Cd300a−/− BMMCs before and after stimulation of FcεRI (see Fig E2, C). Moreover, even in the low concentrations of BMMCs in the culture, in which trans-interactions of CD300a with PS were unlikely, Cd300a−/− BMMCs still showed increased degranulation compared with WT BMMCs (see Fig E2, D and E). Treatment with a neutralizing anti-CD300a antibody (EX42) increased CD107a expression on WT BMMCs to a level comparable to that of Cd300a−/− BMMCs (Fig 2, D). Similarly, a neutralizing anti-human CD300a antibody increased the CD107a expression during FcεRI-mediated degranulation of hMCs (Fig 2, E). Together, these results further support the involvement of cis-interaction, rather than trans-interaction, of CD300a with PS in the suppression of MC degranulation. In contrast, CD107a expression was comparable between WT and Cd300a−/− BMMCs stimulated with either ATP or ionomycin (see Fig E2, F). Interestingly, FcεRI stimulation also induced polarization of FcεRI and colocalization of FcεRI with CD300a and PS (see Fig E2, G and H). Thus, the same spatiotemporal localization of CD300a, PS, and FcεRI on MCs might cause CD300a-mediated suppression of FcεRI-mediated, but not ATP- or ionomycin-mediated, CD107a expression during degranulation. Together, these imaging and functional analyses suggest that the cis-interaction of CD300a with PS specifically suppresses FcεRI-mediated signaling for degranulation of MCs. Indeed, Syk phosphorylation was higher in Cd300a−/− BMMCs than in WT BMMCs after antigen challenge at 10 minutes (600 seconds) after FcεRI stimulation, but was comparable between WT and Cd300a−/− BMMCs at 2 minutes (120 seconds) after stimulation (see Fig E3 in this article's Online Repository at www.jacionline.org), consistent with our observation that degranulation was significantly higher in Cd300a−/− than in WT BMMCs at 8 minutes (480 seconds), but not 2 minutes (120 seconds), after FcεRI stimulation (Fig 2, B). We further analyzed the role of CD300a in the pathogenesis of passive systemic anaphylaxis (PSA). WT and Cd300a−/− mice were intravenously injected with TNP-specific IgE, followed by intravenous challenge with TNP-OVA. Although rectal temperature of WT and Cd300a−/− mice decreased to a similar level by 20 minutes (1200 seconds) after the challenge, the recovery of the rectal temperature after this period was slower in Cd300a−/− mice than in WT mice (Fig 2, F). Interestingly, this seems to correlate to the time course of the self-regulation of MCs degranulation in vitro by CD300a. Similar results were also observed when mice were intraperitoneally injected with a neutralizing anti-CD300a antibody (EX42) (Fig 2, G), suggesting that the CD300a-PS interaction suppressed PSA. Moreover, polarized PS was detected only in MCs, rather than at regions surrounding MCs, in the ear tissue of mice after injection with Flag-tagged MFG-E8-D89E (milk fat globule-EGF factor 8 protein- D89E) together with TNP-OVA (see Fig E4 in this article's Online Repository at www.jacionline.org). These results suggest that the cis-interaction regulated MCs degranulation in vivo as well as in vitro. In conclusion, by combining imaging and functional analyses of MC degranulation, we revealed the physical and functional associations of externalized PS with CD300a via cis-interaction on viable MCs during degranulation (see Fig E5 in this article's Online Repository at www.jacionline.org). Given that MCs are widely distributed in many tissues in a scattered manner without contact with each other7Irani A.A. Schechter N.M. Craig S.S. DeBlois G. Schwartz L.B. Two types of human mast cells that have distinct neutral protease compositions.Proc Natl Acad Sci USA. 1986; 83: 4464-4468Crossref PubMed Scopus (796) Google Scholar and apoptotic cells,8Poon I. Lucas C. Rossi A. Ravichandran K. Apoptotic cell clearance: basic biology and therapeutic potential.Nat Rev Immunol. 2014; 14: 166-180Crossref PubMed Scopus (757) Google Scholar such self-regulation of MC degranulation represents a novel strategy that MCs evolved to control their own activation, adding another layer of regulation in allergic responses. We thank S. Tochihara and W. Saito and the member of the Shibuya Laboratory for secretarial assistance and comments and discussions, respectively. BMMCs were generated by culturing WT and Cd300a−/− mouse bone marrow cells in the presence of 10 ng/mL stem cell factor (455-MC/CF, R&D Systems, Minneapolis, Minn) and 4 ng/mL IL-3 (403-ML, R&D Systems) as previously described.E4Joulia R. L'Faqihi F.-E. Valitutti S. Espinosa E. IL-33 fine tunes mast cell degranulation and chemokine production at the single-cell level.J Allergy Clin Immunol. 2017; 140: 497-509.e10Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Briefly, weekly passages were performed by seeding 2 × 106 cells in 10 mL medium. Cells were cultured for 5 to 8 weeks before use. Cd300a−/− mice were described previously.E4Joulia R. L'Faqihi F.-E. Valitutti S. Espinosa E. IL-33 fine tunes mast cell degranulation and chemokine production at the single-cell level.J Allergy Clin Immunol. 2017; 140: 497-509.e10Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Cultured human synovial MCs were prepared and maintained as previously reported.E5Lee H. Kashiwakura J. Matsuda A. Watanabe Y. Sakamoto-Sasaki T. Matsumoto K. et al.Activation of human synovial mast cells from rheumatoid arthritis or osteoarthritis patients in response to aggregated IgG through Fcγ receptor I and Fcγ receptor II.Arthritis Rheum. 2013; 65: 109-119Crossref PubMed Scopus (41) Google Scholar All mice experiments were conducted in accordance with the guidelines of the Animal Ethics Committee of the University of Tsukuba Animal Research Center. The human mast cell study was approved by the Ethics Committee of the Nihon University School of Medicine (RK-160112-2), and all the subjects provided written informed consent in accordance with the Helsinki Declaration of the World Medical Association. Anti-mouse CD107a (1D4B), anti-mouse c-Kit (2B8), anti-mouse IgE (RME-1), mouse IgG1 (MOPC-21), anti-human CD107a (H4A3), and anti-Flag (L5) antibodies and fluorescein isothiocyanate (FITC)-Avidin were purchased from Biolegend (San Diego, Calif). Human IgE Myeloma (401152) was purchased from CALBIOCHEM. Anti-human IgE (Dε2) (E124.2.8) was purchased from Beckman Coulter (Brea, Calif). Annexin V, trinitrophenyl (TNP)-specific mouse IgE (C38-2) was purchased from BD Bioscience (Franklin Lakes, NJ). Anti-Syk (#2712) and anti-pSyk (#2711) antibodies were purchased from Cell Signaling Technology (Danvers, Mass). TNP-OVA, MFG-E8-D89E, MFG-E8-EPT, neutralizing and non-neutralizing anti-mouse CD300a antibodies (EX42 and TX10, respectively), CD300a-human Fc protein and antihuman CD300a (mouse IgG1, TX49) were made in our laboratory, as previously described.E6Yotsumoto K. Okoshi Y. Shibuya K. Yamazaki S. Tahara-Hanaoka S. Honda S. et al.Paired activating and inhibitory immunoglobulin-like receptors, MAIR-I and MAIR-II, regulate mast cell and macrophage activation.J Exp Med. 2003; 198: 223-233Crossref PubMed Scopus (86) Google Scholar, E7Nakahashi-Oda C. Tahara-Hanaoka S. Honda S. Shibuya K. Shibuya A. Identification of phosphatidylserine as a ligand for the CD300a immunoreceptor.Biochem Bioph Res Commun. 2012; 417: 646-650Crossref PubMed Scopus (55) Google Scholar 1-Oleoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoserine (NBD-PS) was from Avanti. LPS (L2880) derived from Escherichia coli O55:B5, ATP (A26209), and ionomycin (I9657) were purchased from Sigma-Aldrich (St Louis, Mo). PSVue480 and PSVue643 were purchased from Molecular Targeting Technologies (West Chester, Pa). Recombinant mouse IL-33 was purchased from R&D Systems (3626-ML). CD300a-Fc–blocking experiment was done with either apoptotic cell induced by dexamethasone or degranulated BMMCs. Briefly, 105 cells were incubated with CD300a-Fc together with 50 ng anti-CD300a antibody (TX10 or EX42) or 100 ng MFG-E8-D89E on ice for 15 minutes, and further stained by anti-CD107a (for BMMCs only) and anti-human IgG Fc-PE (M1310G05, Biolegend) and phycoerythrin (PI). Flow cytometry analyses were performed using an LSRFortessa system (BD Bioscience), and data were analyzed by using FlowJo software (BD Bioscience). For mouse in vitro stimulations, MCs were sensitized with 1 μg/mL TNP-specific IgE overnight, washed twice with Tyrode's buffer, preincubated with reagents as indicated for 30 minutes, and then challenged with 1 ng/mL TNP-OVA for 30 minutes or indicated time point. β-Hexosaminidase activity was measured as previously reported.E8Hitomi K. Tahara-Hanaoka S. Someya S. Fujiki A. Tada H. Sugiyama T. et al.An immunoglobulin-like receptor, Allergin-1, inhibits immunoglobulin E–mediated immediate hypersensitivity reactions.Nat Immunol. 2010; 11: 601Crossref PubMed Scopus (55) Google Scholar Briefly, 50 μL of the culture supernatant was mixed with 50 μL of 4-nitorophenyl-N-acetyl-β-d-glucosaminide (1.3 mg/mL; Sigma, N9376) in substrate buffer (0.4 M citric acid, 0.2 M NaH2PO4, pH 4.5) and incubated at 37°C for 3 hours. The reaction was stopped by adding 100 μL of glycine (0.2 M, pH 10.7), the solution was thoroughly mixed, and then absorbance of 415 nm was determined. CD107a was measured by flow cytometry after staining with anti-cKit and anti-CD107a antibodies and PI. Apoptotic thymocytes were induced in 1 μM dexamethasone with 107 cell/mL RPMI medium for 12 hours. Cell density pictures were acquired under 20× objective lens under bright field and converted into binary by ImageJ. For human MC stimulation, cultured human synovial MCs were sensitized with 500 ng/mL human IgE, stained with 50 μg/mL anti-human CD300a or isotype control antibody, challenged with 300 ng/mL anti-human IgE antibody for 30 minutes, followed by staining with anti-human CD107a and PI, and analyzed by flow cytometry. For measuring PS exposure of BMMCs after stimulations by LPS (1 μg/mL), IL-33 (150 ng/mL), TNP-OVA (10 ng/mL), ATP (0.5 mM), or ionomycin (2500 ng/mL), the cells were incubated with indicated reagents for 20 minutes after IgE sensitization and then stained with antibody against CD107a, annexin V, and PI. For measuring degranulation after ATP (0.5 mM) or ionomycin (500 ng/mL) stimulation, the stimulation time was 30 minutes. For time-lapse imaging, BMMCs were sensitized as mentioned above and then incubated in 500 μL of Tyrode's buffer containing 1 mM PSVue-643 fluorescent probe (Polysciences, Inc, Warrington, Pa) in a glass-bottom dish (CELLview, Greiner Bio-One, Kremsmünster, Austria) for 30 minutes before gently adding 10 μL of TNP-OVA (100 ng/mL). Cells were monitored under a laser scanning confocal microscope (Olympus FV10i) at 10-second intervals under 60× optical magnification. Data were analyzed and exported by FV10-ASW (Olympus, Tokyo, Japan). The video and montage were generated by ImageJ software (NIH, Bethesda, Md) with 10 frames/s. The confocal images of single mast cells were also collected and analyzed on the same platform with PS stained by PSVue480 and CD300a by Alexa647-conjugated anti-CD300a. For imaging flow cytometry, the Image Stream Mark II system (Amnis) was used to observe single cells after 15-minute degranulation and staining. Data were acquired and analyzed by using the Inspire and Ideas software packages (Merck, Kenilworth, NJ), respectively. The Bright Detail Similarity R3 Feature (based on Pearson correlation coefficient) was adopted as the localization measurement according to the manufacturer's instructions. Briefly, the Bright Detail Similarity R3 Feature value was calculated with a customized imaging mask to identify aggregation of the molecule of interest (CD107a or PS) on the cell surface. To detect colocalization of CD107a and PS, the mask was set on the CD107a channel with the following parameters: Threshold (M05, Ch05, 73) and Peak (M05, Ch05, Bright 10). To detect colocalization of CD300a and PS, the mask was set on the PS channel with following parameters: Threshold (M05, Ch05, 60) and Peak (M05, Ch05, Bright 4). The data were exported as a Flow Cytometry Standard file and analyzed by FlowJo (BD Bioscience). FRET analysis was used as direct evidence of cis-interaction between other receptors.E9Masuda A. Nakamura A. Maeda T. Sakamoto Y. Takai T. Cis binding between inhibitory receptors and MHC class I can regulate mast cell activation.J Exp Med. 2007; 204: 907-920Crossref PubMed Scopus (74) Google Scholar To analyze the cis-interaction between CD300a and PS, TNP-specific IgE-sensitized BMMCs were first stained by nonblocking anti-CD300a antibody TX10 (Alexa546 conjugated) for 15 minutes and then labeled by 500 nM NBD-PS in HBSS with 1 mM CaCl2 for 8 minutes in room temperature. The cells were immediately washed by 5 mg/mL fatty acid-free BSA in HBSS with 1 mM CaCl2. The stained cells were challenged by 10 ng/mL TNP-OVA and observed under live imaging conditions with laser set 471 nm/559 nm and filter set 490 to 540 nm and 570 to 620 nm. FRET efficiency was calculated by sensitized emission according to the FRET package instructions in FV10-ASW (Olympus).Efficiency(E)=1−ee+PFRET∗(ΨddΨaa)∗(QdQa)where Ψdd,Ψaa are the collection efficiency in donor and acceptor channel, respectively; Qd,Qaare quantum yield of the donor and acceptor, respectively.PFRET=f−DSBT−ASBTf : is acceptor with donor excitation.DSBT=(ba)∗eASBT=(cd)∗ge is donor with donor excitation; g : is acceptor with acceptor excitation; and e, f, and g were obtained using donor and acceptor double-stained samples (FRET samples). Donor spectral bleedthrough (DSBT) and acceptor spectral bleedthrough (ASBT) were calculated by images from the donor/acceptor single- stained sample excited by designated laser according to the package instruction to obtain the value a, b, c, and d. FRET+ cell percentage was calculated by counting the FRET+ cells in each field. WT and Cd300a−/− mice (age-matched 8- to 14-week-old females) were sensitized by intravenous (IV) administration of 5 μg of TNP-specific IgE (BD Biosciences, C38-2) for 24 hours and then IV challenged with 40 μg TNP-OVA. Body temperature was measured intrarectally at the indicated time points. For antibody blocking, a neutralizing anti-CD300a or isotype (400 μg/mice) was injected intraperitoneally 5 hours before antigen challenge. For staining ear tissue sections, 50 μg/mice MFG-E8-D89E (Flag-taged) were IV injected together with or without 40 μg/mice antigen. Ear tissue was harvested 10 minutes after injection and fixed with formalin. Paraffin sections were deparaffinized, antigen retrieval was done by using AR6 buffer (PerkinElmer, Waltham, Mass), staining for MCs by using FITC-Avidin (Biolegend), and PS by using PE-anti-Flag (L5, Biolegend). One hundred thousand BMMCs were degranulated as described above and immediately washed with ice-cold PBS at the indicated time points. Cells were lysed with 1% (wt/vol) NP40 in the presence of 1mM sodium orthovanadate. The lysates were immunoblotted with antibodies against Syk or phosphorylated Syk. Statistical analyses were performed using GraphPad Prism software (GraphPad Software). For comparing between 2 groups, statistical significance was determined by 2-tailed unpaired Student t test with or without correction by Holm-Sidak method. For comparing more than 2 groups, statistical significance was determined by 2-way ANOVA multiple comparisons with Bonferroni or Sidak test. Error bars indicate SEM unless otherwise mentioned.Fig E2A and B, WT and Cd300a−/− BMMCs were equally mixed and degranulated by IgE-antigen complex and analyzed by flow cytometry. Data are representative plots (Fig E2, A) and the mean CD107a expression (Fig E2, B) in each genotype. C, PI+ cell before and after degranulation in WT and Cd300a−/− BMMCs. D and E, WT and Cd300a−/− BMMCs were diluted into different density (scale 50 μm) (Fig E2, D), and then CD107a+ cells were measured after degranulation in PI- gated cells (Fig E2, E). F, WT and Cd300a−/− BMMCs were stimulated with 0.5 mM ATP or 500 ng/mL ionomycin and analyzed by flow cytometry. G and H, Imaging flow cytometry of naive or antigen-stimulated BMMCs stained with anti-IgE and anti-CD300a antibodies (Fig E2, E), or PSVue643 plus either anti-IgE or anti-cKit (Fig E2, H). Data are representative of 2 (Fig E2, D-H) to 3 (Fig E2, A-C) independent experiments. APC, Allophycocyanin; FSC-A, forward scatter-area; PE, phycoerythrin; PE-Cy7, phycoerythrin-cyanin7; SSC-A, side scatter area.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Western blot analysis of Syk phosphorylation in whole-cell lysates of degranulated WT and Cd300a−/− BMMCs at indicated time points. Data are representative of 2 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Immunohistochemistry analysis of PS exposure in MCs during PSA. WT mice were sensitized with TNP-specific IgE 24 hours before injection of 50 μg MFG-E8-D89E (Flag-taged) together with or without 40 μg TNP-OVA antigen. Ten minutes after the injection, ear tissue sections were stained for MCs by FITC-conjugated Avidin and for PS by PE-conjugated anti-Flag antibody. Scale bar: 50 μm (tissue view) and 5 μm (enlarged cell view). FITC, Fluorescein isothiocyanate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5Graphical summary of this study.View Large Image Figure ViewerDownload Hi-res image Download (PPT) https://www.jacionline.org/cms/asset/748f14cb-7719-4cb9-8bac-33470e2225a3/mmc1.mp4Loading ... Download .mp4 (3.92 MB) Help with .mp4 files Video E1Time-lapse video of Fig 1, A. BMMCs were sensitized by anti-TNP IgE, seeded in Tyrode's buffer containing PSVue 643 with gentle addition of TNP-OVA under monitoring by confocal microscopy (the images at 10-second intervals). The speed of the video is 10 frames/s. Scale: 10 μm.