Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

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 research^1,2,3,4,5. 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 level^6,7. These include the rigidities of subcellular structures or even whole cells^8,9,

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 bovine serum (FBS) using glass slides and remove aggregates and debris by passing the cell suspension through a 70 μm mesh nylon strainer.
   <....

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.......

Materials

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	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