Tag: afm tips

Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels

Representing a central element of the innate immune system, neutrophils are recruited from the blood stream to a site of inflammation. The recruitment process follows a well-defined sequence of events including adhesion to the blood vessel walls, migration, and chemotaxis to reach the inflammatory focus. A common feature of the underlying signalling pathways is the utilization of Ca2+ ions as intracellular second messengers. However, the required Ca2+ influx channels are not yet fully characterized.*

In the article “Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels” Otto Lindemann, Jan Rossaint, Karolina Najder, Sandra Schimmelpfennig, Verena Hofschröer, Mike Wälte, Benedikt Fels, Hans Oberleithner, Alexander Zarbock and Albrecht Schwab report a novel role for TRPC6, a member of the transient receptor potential (TRPC) channel family, in the CXCL1-dependent recruitment of murine neutrophil granulocytes.*

The authors describe how they tested whether TRPC6 channels are central elements of the signalling cascade underlying CXCR2-mediated neutrophil recruitment. They combined intravital microscopy, single-cell force spectroscopy with atomic force microscopy, Ca2+ imaging, and microfluidic flow chamber assays to investigate the role of TRPC6 channels in murine neutrophils for their recruitment in renal ischemia-reperfusion and cremaster models as well as in in vitro assays.*

The study reveals that TRPC6 channels in neutrophils are crucial signalling modules in their recruitment from the blood stream in response to CXCL1.*

The single-cell force spectroscopy experiments were performed by using atomic force microscopy (AFM) with NanoWorld Arrow-TL1 tipless cantilevers which were incubated prior to experiments for 30 min in Cell-Tak to make the AFM cantilever sticky for neutrophils.*


Figure 4a from “Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels” by Otto Lindemann et al:
Force-distance curves were obtained by probing bEnd5 cells with the neutrophil-carrying cantilever using 1 nN maximal loading force
NanoWorld Arrow-TL1 Tipless AFM cantilever, single cantilever beam on a silicon support chip
NanoWorld Arrow-TL1
Tipless cantilever,
single cantilever beam on a silicon support chip

*Otto Lindemann, Jan Rossaint, Karolina Najder, Sandra Schimmelpfennig, Verena Hofschröer, Mike Wälte, Benedikt Fels, Hans Oberleithner, Alexander Zarbock and Albrecht Schwab
Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels
Journal of Molecular Medicine volume 98, pages349–360(2020)
DOI: https://doi.org/10.1007/s00109-020-01872-4

Please follow this external link to read the full article: https://link.springer.com/article/10.1007/s00109-020-01872-4

Open Access The article “ Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels “ by Otto Lindemann, Jan Rossaint, Karolina Najder, Sandra Schimmelpfennig, Verena Hofschröer, Mike Wälte, Benedikt Fels, Hans Oberleithner, Alexander Zarbock and Albrecht Schwab is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Nanomorphological and mechanical reconstruction of mesenchymal stem cells during early apoptosis detected by atomic force microscopy

Stem cell apoptosis exists widely in embryonic development, tissue regeneration, repair, aging and pathophysiology of disease. The molecular mechanism of stem cell apoptosis has been extensively investigated.*

However, alterations in biomechanics and nanomorphology have rarely been studied.*

In the research article “ Nanomorphological and mechanical reconstruction of mesenchymal stem cells during early apoptosis detected by atomic force microscopy “ Xuelian Su, Haijing Zhou, Guangjie Bao, Jizeng Wang, Lin Liu, Qian Zheng, Manli Guo and Jinting Zhang establish an apoptosis model for bone marrow mesenchymal stem cells (BMSCs) and investigated in detail the reconstruction of the mechanical properties and nanomorphology of the cells.*

Atomic force microscopy (AFM), scanning electron microscopy (SEM), laser scanning confocal microscopy (LSCM), flow cytometry and Cell Counting Kit-8 analysis were applied to assess the cellular elasticity modulus, geometry, nanomorphology, cell surface ultrastructure, biological viability and early apoptotic signals (phosphatidylserine,PS).*

The results indicated that the cellular elastic modulus and volume significantly decreased, whereas the cell surface roughness obviously increased during the first 3 h of cytochalasin B (CB) treatment. Moreover, these alterations preceded the exposure of biological apoptotic signal PS.*

These findings suggested that cellular mechanical damage is connected with the apoptosis of BMSCs, and the alterations in mechanics and nanomorphology may be a sensitive index to detect alterations in cell viability during apoptosis. The results contribute to further understanding of apoptosis from the perspective of cell mechanics.*

NanoWorld PNP Silicon Nitride AFM probes of the PNP-DB type were used for the single-cell imaging with Atomic Force Microscopy and nanoindentation experiments described in this research article.*

Figure 4 from “Nanomorphological and mechanical reconstruction of mesenchymal stem cells during early apoptosis detected by atomic force microscopy” by Xuelian Su et al.: Surface topography of BMSCs captured by AFM at different times. Columns A–D indicated the height-measurement images, vertical deflection images, three-dimensional images and cross-sectional images, respectively. The bright area was the elevated part of the cell, where the nucleus was located(A,C). The untreated cells adhered well, and their surface was smooth. The texture of the F-actin bundles is clearly visible (B, 0 h). The surface of treated cells became increasingly rough, the periphery of the cells became irregular and the area of cell extension gradually decreased (A and B, 1 h, 3 h, respectively).
Figure 4 from “Nanomorphological and mechanical reconstruction of mesenchymal stem cells during early apoptosis detected by atomic force microscopy” by Xuelian Su et al.:
Surface topography of BMSCs captured by AFM at different times. Columns A–D indicated the height-measurement images, vertical deflection images, three-dimensional images and cross-sectional images, respectively. The bright area was the elevated part of the cell, where the nucleus was located(A,C). The untreated cells adhered well, and their surface was smooth. The texture of the F-actin bundles is clearly visible (B, 0 h). The surface of treated cells became increasingly rough, the periphery of the cells became irregular and the area of cell extension gradually decreased (A and B, 1 h, 3 h, respectively).

*Xuelian Su, Haijing Zhou, Guangjie Bao, Jizeng Wang, Lin Liu, Qian Zheng, Manli Guo and Jinting Zhang
Nanomorphological and mechanical reconstruction of mesenchymal stem cells during early apoptosis detected by atomic force microscopy
Biology Open (2020) 9, bio048108.
DOI: 10.1242/bio.048108

Please follow this external link to read the full article: https://bio.biologists.org/content/biolopen/9/3/bio048108.full.pdf

Open Access The article “ Nanomorphological and mechanical reconstruction of mesenchymal stem cells during early apoptosis detected by atomic force microscopy “ by Xuelian Su, Haijing Zhou, Guangjie Bao, Jizeng Wang, Lin Liu, Qian Zheng, Manli Guo and Jinting Zhang is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Pectin Interaction with Immune Receptors is Modulated by Ripening Process in Papayas

Dietary fibers have been shown to exert immune effects via interaction with pattern recognition receptors (PRR) such as toll-like receptors (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptors. Pectin is a dietary fiber that interacts with PRR depending on its chemical structure. Papaya pectin retains different chemical structures at different ripening stages. How this influences PRR signalling is unknown.*

The aim of the article “Pectin Interaction with Immune Receptors is Modulated by Ripening Process in Papayas” by Samira B. R. Prado, Martin Beukema, Eva Jermendi, Henk A. Schols, Paul de Vos and João Paulo Fabi was to determine how ripening influences pectin structures and their ability to interact with TLR2, 3, 4, 5 and 9, and NOD1 and 2.*

Papaya ripening is an enzymatic, biochemically driven process that occurs over a short period of time (five days) and involves the mobilization of pectin and the alteration of its chemical composition.

The authors evaluated the interaction of the water-soluble fractions rich in pectin extracted from unripe to ripe papayas. The pectin extracted from ripe papayas activated all the TLR and, to a lesser extent, the NOD receptors. The pectin extracted from unripe papayas also activated TLR2, 4 and 5 but inhibited the activation of TLR3 and 9.*

During papaya ripening, profound changes in pectin structures lead to differences in the biological effects. The data presented in the paper show that papaya pectin extracted from fruit pulp at different ripening points differently interacted with PRR in a ripening-dependent way. The longer chains of HG from unripe papayas pectin, which were less methyl-esterified, inhibited the activation of TLR3 and 9 and activated TLR2 and 4, in contrast to the ripe papaya’s pectin, which have smaller HG chains with medium methyl esterification thus activating TLR2, 3, 4, 5 and 9.*

This variation may represent new biological features of papaya pectin structures in addition to anticancer activities, possibly creating new and cost-effective approaches to extracting papaya pectin with desirable structural and biological features.*

These findings might lead to selection of ripening stages for tailored modulation of PRR to support or attenuate immunity in consumers.*

The changes in Molecular weight ( Mw ) can also be visualized by Atomic Force Microscopy (see Fig. 1C in the paper.)

The AFM images presented in the paper were acquired in tapping mode using an NanoWorld Pointprobe® NCHR AFM probe with a typical spring constant of 42 N/m and typically 320 kHz resonance frequency. The scan speed and scanning resolution were 0.5 Hz and 512 × 512 points, respectively.*

Figure 1 C from “Pectin Interaction with Immune Receptors is Modulated by Ripening Process in Papayas” by Samira B. R. Prado et al. 2020:
(C) Representative topographical AFM images of Un-1-WSF and R-2-WSF. White arrow indicates linear structures, black arrow aggregates and grey arrow the smaller structure from the R-2-WSF. Un-1-WSF: unripe – papaya from 1st day after harvest – water-soluble fraction; Un-2-WSF: unripe – papaya from 2nd day after harvest – water-soluble fraction; I-WSF: intermediate ripening time point – papaya from 3rd day after harvest – water-soluble fraction; R-1-WSF: ripe – papaya from 4th day after harvest – water-soluble fraction; R-2-WSF: ripe – papaya from 5th day after harvest – water-soluble fraction. Please have a look at the full article for the full figure.

*Samira B. R. Prado, Martin Beukema, Eva Jermendi, Henk A. Schols, Paul de Vos and João Paulo Fabi
Pectin Interaction with Immune Receptors is Modulated by Ripening Process in Papayas
Nature Scientific Reports volume 10, Article number: 1690 (2020)
DOI: https://doi.org/10.1038/s41598-020-58311-0

Please follow this external link to read the full article https://rdcu.be/b3Fnb .

Open Access The article “ Pectin Interaction with Immune Receptors is Modulated by Ripening Process in Papayas “ by Samira B. R. Prado, Martin Beukema, Eva Jermendi, Henk A. Schols, Paul de Vos and João Paulo Fabi is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.