Biomembrane Force Probe to Quantitate Receptor-Ligand Interactions

Abstract

Source: Chen, Y., et al., Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell. J. Vis. Exp. (2015)

In this video, biomembrane force probes (BFPs) are used to measure the forces between receptors and ligands. This technique can detect interactions by monitoring the pressure needed to break the bonds between the pMHC conjugated on the probe beads and the TCRs on the target cells.

Protocol

1. Human RBCs Isolation, Biotinylation, and Osmolarity Adjustment

NOTE: Step 1.1 should be performed by a trained medical professional such as a nurse, with an Institutional Review Board-approved protocol.

1. Obtain 8-10 µl (one drop) of blood from a finger prick and add it to 1 ml of the carbonate/bicarbonate buffer (Material list). Gently vortex or pipette the mixture and centrifuge for 1 min at 900 x g. Discard the supernatant and wash once more.
2. In a small beaker weigh 3.5-4 mg of Biotin-PEG3500-NHS linker (Material list). Dissolve it i....

Results

Figure 1: fBFP assembly.

(A) An overview picture of the fBFP hardware system. (B) A schematic drawing of the fBFP hardware system. (C) The dual-cam system “DC2” (or.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium Phosphate Monobasic Monohydrate (NaH<sub>2</sub>PO<sub>4</sub> &bull; H<sub>2</sub>O)	Sigma-Aldrich	S9638	Phosphate buffer preparation
Anhy. Sodium Phosphate Dibasic (Na<sub>2</sub>HPO<sub>4</sub>)	Sigma-Aldrich	S7907	Phosphate buffer preparation
Sodium Carbonate (Na<sub>2</sub>CO<sub>3</sub>)	Sigma-Aldrich	S2127	Carbonate/bicarbonate buffer preparation
Sodium Bicarbonate (NaHCO<sub>3</sub>)	Sigma-Aldrich	S5761	Carbonate/bicarbonate buffer preparation
Sodium chloride (NaCl)	Sigma-Aldrich	S7653	N2-5% buffer preparation
Potassium chloride (KCl)	Sigma-Aldrich	P9541	N2-5% buffer preparation
Potassium phosphate monobasic (KH<sub>2</sub>PO<sub>4</sub>)	Sigma-Aldrich	P5655	N2-5% buffer preparation
Sucrose	Sigma-Aldrich	S0389	N2-5% buffer preparation
MAL-PEG3500-NHS	JenKem	A5002-1	Bead functionalization
Biotin-PEG3500-NHS	JenKem	A5026-1	RBC biotinylation
Nystatin	Sigma-Aldrich	N6261	RBC osmolarity adjustment
Ammonium Hydroxide (NH<sub>4</sub>OH)	Sigma-Aldrich	A-6899	Glass bead silanization
Methanol	BDH	67-56-1	Glass bead silanization
30% Hydrogen Peroxide (H<sub>2</sub>O<sub>2</sub>)	J. T. Barker	Jan-86	Glass bead silanization
Acetic Acid (Glacial)	Sigma-Aldrich	ARK2183	Glass bead silanization
3-Mercaptopropyltrimethoxysilane (MPTMS)	Uct Specialties, llc	4420-74-0	Glass bead functionalization
Borosilicate Glass beads	Distrilab Particle Technology	9002	Glass bead functionalization
Streptavidin-Maleimide	Sigma-Aldrich	S9415	Glass bead functionalization
BSA	Sigma-Aldrich	A0336	Ligand functionalizing
Fura2-AM	Life Technologies	F-1201	Intracellular calcium fluorescence dye loading
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2650	Intracellular calcium fluorescence dye loading
Quantibrite PE Beads	BD Biosciences	340495	Density quantification
Capillary Tube 0.7-1.0 mm x 30 inches	Kimble Chase	46485-1	Micropipette making
Flaming/Brown Micropipette Puller	sutter instrument	P-97	Micropipette making
Narishige	Narishige	Narishige	Micropipette making
Mineral Oil	Fisher Scientific	BP2629-1	Chamber assembly
Microscope Cover Glass	Fisher Scientific	12-544-G	Chamber assembly
Micro-injector	World Precision Instruments	MF34G-5	Chamber assembly
1 ml syringe	BD	309602	Chamber assembly
Micropipette holder	Narishige	HI-7	Chamber assembly
Microscope (TiE inverted)	Nikon	MEA53100	BFP system
Objective CFI Plan Fluor 40x (NA 0.75, WD 0.72 mm, Spg)	Nikon	MRH00401	BFP system
Camera, GE680, 640 x 480, GigE, 1/3" CCD, mono	Graftek Imaging	02-2020C	BFP system
Prosilica GC1290 - ICX445, 1/3", C-Mount, 1280 x 960, Mono., CCD, 12 Bit ADC	Graftek Imaging	02-2185A	BFP system
Manual submicron probehead with high resolution remote control	Karl Suss	PH400	BFP system
Anti-vibration table (5&rsquo; x 3&rsquo;)	TMC	77049089	BFP system
3D manual translational stage	Newport	462-XYZ-M
SolidWorks 3D CAD software	SolidWorks 3D CAD software	Version 2012 SP5	BFP system
LabVIEW software	National Instruments	Version 2009	BFP system, BFP program
3D piezo translational stage	Physik Instrumente	M-105.3P	BFP system
Linear piezo accuator	Physik Instrumente	P-753.1CD	BFP system
Carbonate/bicarbonate buffer (pH 8.5)			8.4 g/L sodium carbonate (Na2CO3), 10.6 g/L sodium bicarbonate (NaHCO3)
N2-5% buffer (pH 7.2)			27.6 g/L NaPhosphate monobasic (NaH2PO4 &bull; H2O), 28.4 g/L Anhy. NaPhosphate dibasic (Na2HPO4)
&nbsp; &nbsp;	&nbsp; &nbsp;	&nbsp; &nbsp;	20.77 g/L potassium chloride (KCl), 2.38 g/L sodium chloride (NaCl), 0.13 g/L potassium phosphate monobasic (KH2PO4), 0.71 g/L anhy. sodium phosphate dibasic (Na2HPO4), 9.70 g/L sucrose