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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.
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.
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,
The mouse experiment protocol follows the animal care guidelines of Tsinghua University
1. Cantilever functionalization with single T cells
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.......
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.......
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).
....Name | Company | Catalog Number | Comments |
Material | |||
10 μl pipette tip | Thermo Fisher | 104-Q | |
15 ml tube | Corning | 430791 | |
6 cm diameter culture dish | NALGENE nunc | 150462 | |
6-well culture plate | JET | TCP011006 | |
AFM Cantilever | NanoWorld | Arrow-TL1-50 | tipless cantilever |
β-Mercaptoethanol | Sigma | 7604 | |
Biocompatible glue | BD Cell-Tak | 354240 | |
CD4+ T cell isolation Cocktail | STEMCELL | 19852C.1 | |
DC2.4 cell line | A gift from K. Rock (University of Massachusetts Medical School, Worcester, MA) | ||
Dextran-coated magnetic particles | STEMCELL | SV30010 | |
EDTA | GENEray | Generay-E1101-500 ml | |
Epoxy | ERGO | 7100 | |
Ethanol | twbio | 00019 | |
FBS | Ex Cell Bio | FSP500 | |
FcR blocker | STEMCELL | 18731 | |
Glass coverslip | local vender (Hai Men Lian Sheng) | HX-E37 | 24mm diameter, 0.17mm thinckness |
Glass slides | JinTong department of laboratory and equipment management, Haimen | N/A | customized |
H2O2 (30%) | Sino pharm | 10011218 | |
H2SO4 | Sino pharm | 80120892 | |
HEPES | Sigma | 51558 | |
Magnet | STEMCELL | 18000 | |
Mesh nylon strainer | BD Falcon | REF 352350 | |
Moesin-EGFP | N/A | cloned in laboratory | |
Mouse CD25 Treg cell positive isolation kit | STEMCELL | 18782 | Component: FcR Blocker,Regulatory T cell Positive Selection Cocktail, PE Selection Cocktail, Dextran RapidSpheres, |
Mouse CD4+ Tcell isolation kit | STEMCELL | 19852 | Component:CD4+T cell isolation Cocktail, Streptavidin RapidSpheres, Rat Serum |
NaOH | Lanyi chemical products co., LTD, Beijing | 1310-73-2 | |
PBS | Solarbio | P1022-500 | |
PE selection cocktail | STEMCELL | 18151 | |
Penicillin-Streptomycin | Hyclone | SV30010 | |
PLCδ-PH-mCherry | Addgene | 36075 | |
Polystyrene microspheres 6.0μm | Polysciences | 07312-5 | |
polystyrene round bottom tube | BD Falcon | 352054 | |
Rat serum | STEMCELL | 13551 | |
RAW264.7 | Â ATCC | ||
Recombinant Human Interleukin-2 | Peprotech | Peprotech, 200-02-1000 | |
Red blood cell lysis buffer | Beyotime | C3702 | |
Regulatory T cell positive selection cocktail | STEMCELL | 18782C | |
RPMI 1640 | Life | C11875500BT | |
Sample chamber | Home made | ||
Streptavidin-coated magnetic particles | STEMCELL | 50001 | |
Transfection kit | Clontech | 631318 | |
Trypsin 0.25% EDTA | Life | 25200114 | |
Tweezers | JD | N/A | |
Name | Company | Catalog Number | Comments |
Equipment | |||
20x objective NA 0.8 | Zeiss | 420650-9901 | Plan-Apochromat |
Atomic force microscope | JPK | cellHesion200 | |
Centrifuge | Beckman coulter | Allegra X-12R | |
Fluorescence imaging | home-made objective-type total internal reflection fluorescence microscop based on a Zeiss microscope stand | ||
Humidified CO2 incubator | Thermo Fisher | HERACELL 150i | |
Inverted light microscope | Zeiss | Observer A1 manual |
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