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laminin under microscope

Laminin IV domain is not found in short laminin chains (alpha4 or beta3). Our previous work has shown that the basement membrane of vascular endothelium affects leukocyte extravasation by influencing adhesion and migration of extravasating cells (6, 7), and by affecting the tightness of endothelial monolayer (8). Often indicate exsolution-the solid-state separation of a mineral mixture into different components during cooling. Intravital microscopy was performed using an Axioscope A1 microscope equipped with an immersion objective (SW 40/0.75 NA) and stroboscopic epifluorescent illumination (Colibri 2, Zeiss), as described previously (8). 107 bone marrow cells from femurs and tibias of CX3CR1GFP/+ mice were intravenously (i.v.) Endothelial laminin-α5 is dispensable for BBB maintenance under homeostatic conditions but inhibits inflammatory cell extravasation in pathological conditions. mit anderen Laminin-Molekülen Bindungen eingehen können. After live cell imaging the cremaster muscle was removed, fixed in 1% PFA for 1 h, blocked and permeabilized in PBS containing 5% FCS and 0.5% Triton X-100 for 30 min. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Furthermore, laminin-332, which was differentially expressed in keratinocytes versus melanoma cells , was found to be crucial for the attachment and spreading of melanoma cells on the keratinocyte-derived ECM (Figs. As seen by dual-label immunofluorescence microscopy in Figure ... Studies examining the effects of laminin-10 delivery to later stages of skin development are currently under way. However, the number of extravasated GFP+ monocytes at 4 h after CCL2 application was significantly lower in Lama4−/− hosts compared to WT hosts (Figure 1B). Hey, this salami looks like Slash! Human monocytes were isolated from buffy coats of blood from healthy donors. Play Download. Monocyte purity was >85% as defined by flow cytometry for CD14 antibody (M5E2, Biolegend). The localization of the extracellular matrix components laminin, fibronectin and type IV collagen in the enteric nervous system and the surrounding smooth muscle was investigated by immunohistochemical methods, using tissue sections of rat and guinea pig large intestine. We therefore transferred bone marrow cells from CX3CR1GFP/+ transgenic mice to Lama4−/− and Tek-cre::Lama5−/− hosts and their corresponding WT littermates to generate bone marrow chimeras for in vivo tracking of monocytes and specifically induced monocyte extravasation by intra-scrotal injection of CCL2 (27). Cells were collected by 0.05% trypsin treatment and processed according to the recom- Cougoule C, Van Goethem E, Le Cabec V, Lafouresse F, Dupre L, Mehraj V, et al. The temperature was maintained at 18–23°C and humidity at 40–70%. Science (2007) 317(5838):666–70. 13. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Nat Immunol (2014) 15(10):929–37. 30. Laminin‐221 improved tolerance to hypoxia of human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPS‐CMs). Monocytes were cultured overnight in McCoy’s medium (Sigma-Aldrich) plus 15% FCS at 37°C and 7% CO2 in Teflon cell culture bags (OriGen) prior to use. Laminin-5 (also known as laminin-332, kalinin, nicelin, epiligrin, and radsin), which contains α3, β3, and γ2 chains, is one of the BM components secreted by epithelial cells themselves, whereas integrins play central roles in the adhesion of cells to the BM and subsequent signal transduction across the plasma membrane (Hintermann and Quaranta, 2004; Katayama and Sekiguchi, 2004). Mol Cell Biol (1994) 5:679–90. LoG detector was used to detect the CX3CR1-GFP cells and Simple LAP tracker was used for tracking the cells. Front. Immunity (2003) 19(1):71–82. This work was supported by funding from the German Research Foundation (CRC1009A02, B08, B09, and EXE1003). The three short arms of the molecule are particularly efficient at binding with other similar molecules, thus forming sheets that make up basement membranes while the fourth, longer arm can bind with cells. Endothelial cells (e.END4.1, e.ENDwt) were seeded onto the collagen type I gel base and were incubated at 37°C overnight. However, it can be seen that the evidence is presented in a particular fashion so that the audience will be more likely to agree with the evangelist. The niche is a specialized microenvironment for tissue stem cells in vivo. Several studies have demonstrated that the endothelium plays a role in the switch to macrophages (1, 5); whether the underlying endothelial basement membrane also contributes to this step has not been considered to date. Cells were fixed after 15 days and stained with GATA-4 and nestin antibodies. Due to the difficulty in obtaining sufficient numbers of monocytes for in vitro analyses, there is a paucity of information on monocyte-extracellular matrix interactions relevant to the extravasation process (13). Adhesion assays were carried out using human monocytes and mouse monocyte-like Hoxb8 cells. Correlation of CX3CR1-GFP MFI with speeds of cell migration, revealed that CX3CR1-GFPlow monocytes had higher average speeds of migration than CX3CR1-GFPhigh monocytes (Figure S2B and Figure 2C), as expected for extravasating cells. Image analyses of α3 laminin under stretching. LL carried out the intravital microscopy (IVM) experiments and most adhesion and migration assays. J Immunol (2010) 184(5):2671–6. However, differentiation toward the macrophage lineage is also associated with changes in size and cytoplasmic complexity (53) that will affect migration potential and speed. Total numbers of cells isolated in adult or P21 colons were the same regardless of genotype. doi: 10.1161/ATVBAHA.109.187450, 29. doi: 10.15252/embj.201694756, 17. To test integrin involvement in binding to the laminins, function blocking antibodies to integrins α6, β1, or β3 were employed in adhesion assays, revealing complete inhibition of mouse Hoxb8 and human monocytes binding to laminin 511 by anti-integrin β1 and α6 antibodies (Figures 3D, E). Laminin is defined by the Webster Medical Dictionary as a “glycoprotein that is a component of connective tissue basement membrane and that promotes cell adhesion.” In other words, looking at laminin as a kind of glue isn’t far from the truth. Eur J Cell Biol (2012) 91(11-12):938–49. <3 Samus M, Seelige R, Schafer K, Sorokin L, Vestweber D. CD99L2 deficiency inhibits leukocyte entry into the central nervous system and ameliorates neuroinflammation. The animal study was reviewed and approved by Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhein-Westfalen; Aktenzeichen 81-02.04.2018.A392. This confirmed overlap between GFP+ and Gr-1+ cells with the exception of few GFP-negative/Gr-1+ cells that represent neutrophils (Figure S1A). doi: 10.1074/jbc.M010155200, 53. Data are means ± SEM from three experiments (marked in different colors) performed with 23 WT, 9 Lama4−/−, and 7 Tek-cre::Lama5−/− mice (marked by different symbols). Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Cells bound to the dots of different substrates were imaged using an inverse Axiovert microscope (Zeiss) and the number of adherent cells per substrate was quantified using ImageJ software. ).Die Polypeptidketten enthalten mehr als 1.500 Aminosäurebausteine. TV generated WT and CD18−/− Hoxb8 monocyte lines and contributed to writing of the manuscript. Various invasive pathogens attach to host tissues via the extracellular matrix component laminin, the major glycoprotein found within basement membranes. Indeed, it may be serendipitous whether an extravasating monocyte encounters a laminin 511 high or low patch in the postcapillary venule basement membrane, which may determine those cells that differentiate toward the macrophage phenotype and those that do not and can re-enter the circulation (53), placing endothelial laminin 511 in a pivotal position in the switch from monocytes to macrophages. J Immunol (2000) 165:5831–8. Sci Rep (2016) 6:25016. doi: 10.1038/srep25016, 34. Doesn't ring any bells here at all. Adherent cells were counted at 1 h and expressed as number of bound cells/mm2. Presented by Chris Smith, Adam Murphy. (C) Cytokine concentrations in conditioned media of eENDwt or eEND4.1 cells cultured in the presence or absence of splenic monocytes for 16 h, and of splenic monocytes alone. The remaining tissue was incubated in digestion buffer [RPMI 1640, 10% FCS, 1.25 mg/ml collagenase D (Roche), 0.85 mg/ml collagenase V (Sigma-Aldrich), 1 mg Dispase (Gibco), and 30 U/ml DNase (Roche)] for 30–45 min at 37°C with shaking. Here is an electron microscope picture of an actual Laminin molecule. Regulation of cellular interactions with laminin by integrin cytoplasmic domains: A and B structural variants of the a6b1 integrin differentially modulate the adhesive strength, morphology and migration of macrophages. The experiment was repeated three times. Intra-scrotal injection of 500 ng CCL2 (Biolegend) in 0.2 ml saline 3.5 or 1.5 h before cremaster muscle exteriorization was employed to specifically induce monocyte extravasation. Non-adherent cells were removed and attached cells were fixed in 4% paraformaldehyde (PFA) for 10 min and stained by 0.5% crystal violet for 10 min. And what does laminin look like? Heparansulfat; Proteoglykan; Einzelnachweise Diese Seite wurde zuletzt am 27. Monocyte differentiation and antigen-presenting functions. (A) Representative flow cytometry showing proportions of early extravasating P1 (Ly6ChiMHCIIlow) and P2 (Ly6CmidMHCIImid) populations, and mature P3 macrophages (Ly6ClowMHCIIhi) in isolated colonic lamina propria cells from adult WT, Lama4−/− and Tek-cre::Lama5−/− mice; (B) corresponding quantification of the data and (C) ratios of P3/P2 cells. A minimum of 20 randomly selected cells on each substrate were manually tracked and cell velocities were measured/experiment. The cells were incubated for 24 h at 37°C and 5% CO2 on bacterial plates, non-adherent cells were then harvested, transferred to a new bacterial plate in DMEM plus 10% FCS supplemented with 20 ng/ml M-CSF (Promokine) and cultured for 8 days to generate bone marrow derived macrophages (BMDMs). Share. Laminin α5 modulates fibroblast proliferation in epidural ... Stained cells were observed under an optical photographic light microscope at ×200 magnification and analyzed ... system, cell nuclei were stained with Hoechst 33342 for 5 min at room temperature. In reality its conformation is far more convoluted. doi: 10.1016/j.cellimm.2014.03.012, 40. Nat Rev Immunol (2017) 17(6):349–62. Such changes are likely to affect the ability of the cells to interact with and to distinguish between the different laminin isoforms. 1 hour before injection of microspheres. They represent an important early line of defense against infectious agents or reaction to tissue damage and in several tissues bone marrow derived monocytes play a critical role in the replenishment of resident monocytes, macrophages, and dendritic cells (DCs) required for tissue immunity (1). Recorded images were analyzed using ImageJ software. Dynamic in situ cytometry was employed to investigate associations between laminin 511 expression and CX3CR1-GFP mean fluorescence intensity (MFI): cremaster muscles were stained with rat anti-mouse PECAM-1 (MEC13.3, BD Bioscience) and rabbit anti-mouse laminin α5 (antibody 405) (6) and monocytes were identified by CX3CR1-GFP expression; Alexa 647–conjugated anti-rat and Alexa 555–conjugated anti-rabbit secondary antibodies (Abcam) were employed. Postcapillary venules of the colon wall showed the same patchy laminin α5 and even laminin α4 distribution as occurs in other tissues (Figure S6B). Endothelioma cells expressing the polyoma middle T oncogene induce hemangiomas by host cell recruitment. *Correspondence: Lydia Sorokin,, †These authors have contributed equally to this work, Front. Cytokines were measured in 5× concentrated conditioned media using Th1/Th2 10 Plex Flowcytomix Kit (eBioscience). In normal mice, integrin alpha6beta4, a receptor for laminin alpha5, is strongly localized at the basal layer of the epithelium, whereas in mutant mice, integrin alpha6beta4 is expressed around the cell surface. Significance was calculated using a Mann-Whitney test; exact p-values are shown in the graph. Laminin. Typically, 4–5 million single cells were obtained from one adult colon, and approximately 2 million single cells from one P21. [5], Besides, if you look at it under an electron microscope (provided, of course that you're A) not a fundie and B) haven't been buttered up into accepting anything as cross shaped by a pastor first) it looks more like a pretty damn awesome shape for a sex toy. The experiments with adult and P21 mice were done three times with at least two mice/mouse strain/experiment. Rats were sacrificed by decapitation and the outer membrane of the sciatic nerve was gently removed under a microscope (Leica Microsystems GmbH, Wetzlar, Germany; magnification, ×10), and ophthalmic scissors were used to cut the outer membrane into pieces. As it is not possible to isolate sufficient leukocytes from the cremaster muscle to phenotype the extravasated GFP+ cells, we investigated the intestine where resident macrophages of the lamina propria (LP) are constantly replenished by circulating monocytes (36) and extravasate across postcapillary venules. These experiments were repeated three times with triplicates/experiment. Flow cytometry analyses of the main laminin binding integrins, α6 and αv, on P1, P2, and P3 populations in adult and P21 LP, revealed higher levels of integrin αv in P3 macrophages (Figure 4F) as also observed in BMDM (Figure S3C); however, integrin α3 was not detectable on P1, P2 or P3 populations from adult or P21 tissues. Laminin is a protein that is part of the extracellular matrix in humans and animals. Experiments were performed using age and sex matched WT chimera and laminin knockout chimeras. Feb 26, 2012 - Laminin is the cell adhesion molecule that makes up our bodies. CX3CR1-GFP MFI was measured in areas of high and low laminin 511 expression using dynamic in situ cytometry, confirming a higher proportion of CX3CR1-GFPlow cells at laminin 511low than at laminin 511high sites, and correspondingly higher proportions of CX3CR1-GFPhigh monocytes at laminin 511high sites (Figure 2B and Figure S2A). Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. Application of the “two-slit” photometric technique to the measurement of microvascular volumetric flow rates. S2A), revealing a higher proportion of CX3CR1-GFPlow cells, expressed as a percent of total GFP+ cells, at laminin 511low compared to laminin 511high sites; data are means ± SD of three mice from three separate experiments and at least five postcapillary venules in two different areas/mouse; different experiments are marked by different colors. Blood leukocytes and macrophages of various phenotypes have distinct abilities to form podosomes and to migrate in 3D environments. The basal lamina helps to attach and anchor the cells to the underlying connective tissue. Quantification of adherent and extravasating CX3CR1-GFP+ monocytes at (B) 4 h and/or (C) 2 h post-application of CCL2. Recent intravital imaging of sterile inflammation in the cremaster muscle also revealed that extravasating myeloid cells spend disproportionately long periods of time at the interface between the endothelial monolayer and the endothelial basement membrane and in breaching the basement membrane compared to their rapid penetration of the endothelial monolayer (8), suggesting that the subendothelial compartment may be a site of reprogramming required for subsequent steps (10). And in some diagrammatic representations of laminin, the ‘arms’ of the cross, i.e. the B … (C) Quantification of GFP MFI and migration speed of individual CX3CR1high and CX3CR1low cells analyzed in three WT hosts from three separate experiments. A two-step procedure with a single gradient in each step was used, which included centrifugation through a Pancoll gradient (Pan-Biotech) (density = 1.070 g/ml) followed by a Percoll (General Electric Company) gradient (density = 1.064 g/ml). The adult bone marrow retains two populations of stem cells with emerging importance for the treatment of diverse liver diseases: hematopoietic stem c… Colonic LP samples (2 million cells/staining) were stained at 4°C for 20 min with the following antibodies: Ly6C-FITC (AL-21, BD Pharmingen), F4/80-PE (BM8, BioLegend), Siglec F-PE-CF594 (E50-2440, BD Bioscience), Ly6G PE-Cy7 (1A8, BioLegend), CD11b-APC (M1/70, eBioscience), CD11c-AF700 or -APC-R700 (N418, BioLegend), MHCII-BV421 (M5/114.15.2, BioLegend), and CD45-BV510 (30-F11, BioLegend) at 4°C for 20 min. Endothelial basement membranes of arteries, arterioles and capillaries typically show high expression of the two main endothelial laminins, laminin 411 and 511, with progressively less laminin 511 occurring in the postcapillary venules, venules and veins resulting in patches of laminin 411 positive and laminin 511 low expression which define the preferred sites of leukocyte extravasation (6, 7, 9). For some experiments, mice were injected with recombinant mouse TNF-α (12 μg/kg i.v.) 1 decade ago. J Cell Sci (2000) 113(5):869–76. One of the chains, Ln-γ2, is a specific marker of invasive tumors because it is frequently expressed as a monomer in malignant tumors. Biological replicates were individual animals or chimeras, technical replicates were analyses performed on different postcapillary venules in the same animal. 1 decade ago. Splenic monocytes were isolated by depleting CD19, Ly6G, CD11c, CD3ε, Siglec-F, MHCII, and CD11c positive cells using magnetic beads (Miltenyi Biotec); 5 × 105 cells were plated onto the confluent endothelial cells for 16 h at 37°C. Lipowsky HH, Zweifach BW. Proc Natl Acad Sci USA (2013) 110(31):E2915–24. To determine laminin β1 versus laminin β2 expressions at the protein level, fluorescence immunostaining was performed in 3 young (12–15 weeks) and 3 aged (18 months) mouse heart cryosections. Cell Immunol (2014) 291(1-2):41–8. Es sind drei verschiedene Lamine mit jeweils verschiedenen Isoformen bekannt: . Without laminin, you’d fall apart. Naïve splenic monocytic cells (Figure S7B) were incubated on confluent monolayers of eENDwt or eEND4.1 for 16 h and cells localised at the subendothelial layer were analyzed by flow cytometry as described previously (5), revealing a significantly higher proportion of MHCII+Ly6Cneg and F4/80+Ly6Cneg macrophages in eEND4.1 than in eENDwt cocultures (Figures 5A, B). Most rational people, hell, just most people, wouldn't be particularly struck by the facts. They gave me a video talk about the molecule Laminin, the speaker (Louie G) said. doi: 10.1073/pnas.90.19.9051, 24. Image by Vanessa Fitzgerald Laminin The ‘cross-shaped’ laminin molecule. This makes me wonder if he intentionally edited out the other image. Sixt M, Engelhardt B, Pausch F, Hallmann R, Wendler O, Sorokin LM. doi: 10.4049/jimmunol.165.10.5831. Our data demonstrate that it is not the endothelium alone but also laminin 511 in the underlying basement membrane that is required for this switch. Poly-L-Ornithine/Laminin. Cloning of the mouse laminin alpha 4 cDNA. doi: 10.1038/nmeth865, 20. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). While several factors such as colony stimulation factor 1 (CSF1) and TGF-β have been shown to play important roles in this switch in the intestine, current data suggests that additional factors must be involved (36, 39). Nat Immunol (2011) 12(8):761–9. doi: 10.1016/j.celrep.2016.12.092, 9. Wynn TA, Chawla A, Pollard JW. Biological replicates were different batches of Hoxb8 cells, human monocytes or BMDM preparations used in separate experiments, technical replicates were repetitions of the same test (e.g., adhesion on a specific laminin isoform) in one experiment. 1 Answer. J Exp Med (1998) 188(1):119–31. However, BMDM migration on laminin 411 and 511 did differ: As macrophages are unlikely to transmigrate across endothelial monolayers and their basement membranes in vivo, we analyzed 2D chemotaxis of BMDM using time-lapse video microscopy, revealing BMDM migration on both laminins 411 and 511 but more extensive and faster migration on laminin 411 compared to laminin 511 … Ultrapure Laminin (entactin-free), sterile filtered. Application. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Wang GG, Calvo KR, Pasillas MP, Sykes DB, Hacker H, Kamps MP. Copyright © 2020 Li, Song, Chuquisana, Hannocks, Loismann, Vogl, Roth, Hallmann and Sorokin. There are several different laminins. Therefore, mouse integrin β2 null (CD18−/−) (20) Hoxb8 monocytes were generated and employed in all subsequent adhesion assays, and human monocytes were treated with anti-human integrin β2 blocking antibody (TS1/18). doi: 10.1111/j.1432-1033.1997.t01-1-00727.x. BioLamina's Biolaminin substrates are the only original full length, recombinant laminins on the market. Like T cells and neutrophils, CX3CR1-GFP+ monocytes also preferentially extravasated at laminin 511low sites, with CX3CR1-GFPlow inflammatory-monocytes showing preferential localization and higher motility at such sites. In (B, C, F, G) each dot or square denotes one mouse and the different experiments are marked by different colors; significance was calculated using Mann-Whitney tests as not all data passed the normality test by D`Agostino & Pearson; the exact p-values are shown in the graphs. 1987 ).r under reducing... Universal microscope equipped with epifluorescence optics flow.. 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