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Characterization of the Elasticity of CD4+ T Cells: An Approach Based on Peak Force Quantitative Nanomechanical Mapping

  • CD4+ T cells are essential players in orchestrating the specific immune response against intracellular pathogens, and in inhibiting tumor development in an early stage. The activation of T cells is triggered by engagement of T cell receptors (TCRs). Here, CD3 and CD28 molecules are key factors, (co)stimulating signaling pathways essential for activation and proliferation of CD4+ T cells. T cell activation induces the formation of a tight mechanical bond between T cell and target cell, the so-called immunological synapse (IS). Due to this, mechanical cell properties, including stiffness, play a significant role in modulating cell functions. In the past, many approaches were made to investigate mechanical properties of immune cells, including micropipette aspiration, microplate-based rheometry, techniques based on deformation during cytometry, or the use of optical tweezers. However, the stiffness of T lymphocytes at a subcellular level at the IS still remains largely elusive.With this protocol, we introduce a method based on atomic force microscopy (AFM), to investigate the local cellular stiffness of T cells on functionalized glass/Polydimethylsiloxan (PDMS) surfaces, which mimicks focal stimulation of target cells inducing IS formation by T cells. By applying the peak force nanomechanical mapping (QNM) technique, cellular surface structures and the local stiffness are determined simultaneously, with a resolution of approximately 60 nm. This protocol can be easily adapted to investigate the mechanical impact of numerous factors influencing IS formation and T cell activation.Graphical abstract: Overview of the experimental workflow.Individual experimental steps are shown on the left, hands on and incubation times for each step are shown right.

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Metadaten
Document Type:Article
Author:Philipp JungORCiD, Xiangda Zhou, Sandra IdenORCiD, Bin QuORCiD, Markus BischoffORCiD
URN:urn:nbn:de:bsz:291:415-467
DOI:https://doi.org/10.21769/BioProtoc.4383
ISSN:2331-8325
Parent Title (English):bio-protocol
Volume:12
Issue:8
First Page:e4383
Language:English
Year of first Publication:2022
Release Date:2022/05/23
Tag:AFM; CD4+ T cell; Peak Force QNM; elasticity mapping; stiffness
Funding Information:Deutsche Forschungsgemeinschaft (SFB 1027 projects A2 to BQ, B2 to MB, A12 to SI, and SPP1782 ID79/2-2 to SI); INM Fellow
Scientific Units:Fellow
DDC classes:500 Naturwissenschaften und Mathematik / 570 Biowissenschaften, Biologie
Open Access:Open Access
Signature:INM 2022/052
Licence (German):License LogoCreative Commons - CC BY - Namensnennung 4.0 International