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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Abstract

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

Introduction

Atomic force microscopy (AFM), a versatile tool, has found many applications in cell biology research1,2,3,4,5. Apart from its high-resolution imaging capability, the native force-probing feature allows biophysical properties of living cells to be investigated directly in situ at the single-cell level6,7. These include the rigidities of subcellular structures or even whole cells8,9,

Protocol

The mouse experiment protocol follows the animal care guidelines of Tsinghua University

1. Cantilever functionalization with single T cells

  1. Mouse spleen cells preparation
    1. Sacrifice the mouse (8-16 weeks of age (either sex); e.g., C57BL/6 strain) using carbon dioxide, followed by cervical dislocation.
    2. Clean the mouse with 75% ethanol and make a midline skin incision followed by splenectomy.
    3. Homogenize the spleen in 4 mL of PBS containing 2% fetal bo.......

Representative Results

Figure 4A shows typical force-distance curves from the binding interaction between single-T cell and single-DC in one approach-retract cycle. The light red curve is the extension curve and the dark red one is the retraction curve. Since the extension curve is typically used for indentation or rigidity-analysis, here only the retraction curve is concerned for cell adhesion. The minimum value (the green circle) in the curve gives a measure of the maximum adhesi.......

Discussion

AFM-based single-cell force spectroscopy has evolved to be a powerful tool to address the biophysical properties of living cells. For those applications, the cantilever needs to be functionalized properly in order to probe specific interactions or properties on the cells of interest. Here, the methods for coupling single T cell and single micron-sized bead to the tip-less cantilever are described respectively. To attach a single T cell to the cantilever, a biocompatible glue was chosen as cell adhesive. It is a specially.......

Acknowledgements

This work is supported by the National Natural Science Foundation of China General Program (31370878), State Key Program (31630023) and Innovative Research Group Program (81621002).

....

Materials

NameCompanyCatalog NumberComments
Material
10 μl pipette tipThermo Fisher104-Q
15 ml tubeCorning430791
6 cm diameter culture dishNALGENE nunc150462
6-well culture plateJETTCP011006
AFM CantileverNanoWorldArrow-TL1-50tipless cantilever
β-MercaptoethanolSigma7604
Biocompatible glueBD Cell-Tak354240
CD4+ T cell isolation CocktailSTEMCELL19852C.1
DC2.4 cell lineA gift from K. Rock (University of Massachusetts Medical School, Worcester, MA)
Dextran-coated magnetic particlesSTEMCELLSV30010
EDTAGENErayGeneray-E1101-500 ml
EpoxyERGO7100
Ethanoltwbio00019
FBSEx Cell BioFSP500
FcR blockerSTEMCELL18731
Glass coversliplocal vender (Hai Men Lian Sheng)HX-E3724mm diameter, 0.17mm thinckness
Glass slidesJinTong department of laboratory and equipment management, Haimen N/Acustomized
H2O2 (30%)Sino pharm10011218
H2SO4Sino pharm80120892
HEPESSigma51558
MagnetSTEMCELL18000
Mesh nylon strainerBD FalconREF 352350
Moesin-EGFPN/Acloned in laboratory
Mouse CD25 Treg cell positive isolation kitSTEMCELL18782Component: FcR Blocker,Regulatory T cell Positive Selection Cocktail, PE Selection Cocktail, Dextran RapidSpheres,
Mouse CD4+ Tcell isolation kitSTEMCELL19852Component:CD4+T cell isolation Cocktail, Streptavidin RapidSpheres, Rat Serum
NaOHLanyi chemical products co., LTD, Beijing1310-73-2
PBSSolarbioP1022-500
PE selection cocktailSTEMCELL18151
Penicillin-StreptomycinHycloneSV30010
PLCδ-PH-mCherryAddgene36075
Polystyrene microspheres 6.0μmPolysciences07312-5
polystyrene round bottom tubeBD Falcon352054
Rat serumSTEMCELL13551
RAW264.7 ATCC
Recombinant Human Interleukin-2PeprotechPeprotech, 200-02-1000
Red blood cell lysis bufferBeyotimeC3702
Regulatory T cell positive selection cocktailSTEMCELL18782C
RPMI 1640LifeC11875500BT
Sample chamberHome made
Streptavidin-coated magnetic particlesSTEMCELL50001
Transfection kitClontech631318
Trypsin 0.25% EDTALife25200114
TweezersJDN/A
NameCompanyCatalog NumberComments
Equipment
20x objective NA 0.8Zeiss420650-9901Plan-Apochromat
Atomic force microscopeJPKcellHesion200
CentrifugeBeckman coulterAllegra X-12R
Fluorescence imaginghome-made objective-type total internal reflection fluorescence microscop based on a Zeiss microscope stand
Humidified CO2 incubatorThermo FisherHERACELL 150i
Inverted light microscopeZeissObserver A1 manual

References

  1. Benoit, M., Gabriel, D., Gerisch, G., Gaub, H. E. Discrete interactions in cell adhesion measured by single-molecule force spectroscopy. Nature Cell Biology. 2 (6), 313-317 (2000).
  2. Chen, J., et al.

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Atomic Force MicroscopeCantilever FunctionalizationSingle T CellSingle ParticleImmunological Force SpectroscopyDendritic Cell T Cell InteractionCell surface InteractionBiocompatible GlueIL 2Sample PreparationCalibrationLiving Cell Environment

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