Biomembrane Force Probe to Quantitate Receptor-Ligand Interactions

Published: June 29, 2023

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. 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 s…

Representative 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” (orange) onto which the high-speed camera (blue) and a fluorescence camera (white) were mounted. (D) The microscope stage that ad…

Divulgazioni

The authors have nothing to disclose.

Materials

Sodium Phosphate Monobasic Monohydrate (NaH2PO4 • H2O) Sigma-Aldrich S9638 Phosphate buffer preparation
Anhy. Sodium Phosphate Dibasic (Na2HPO4) Sigma-Aldrich S7907 Phosphate buffer preparation
Sodium Carbonate (Na2CO3) Sigma-Aldrich S2127 Carbonate/bicarbonate buffer preparation
Sodium Bicarbonate (NaHCO3) 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 (KH2PO4) 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 (NH4OH) Sigma-Aldrich A-6899 Glass bead silanization
Methanol BDH 67-56-1 Glass bead silanization
30% Hydrogen Peroxide (H2O2) 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’ x 3’) 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 • H2O), 28.4 g/L Anhy. NaPhosphate dibasic (Na2HPO4)
            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

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Citazione di questo articolo
Biomembrane Force Probe to Quantitate Receptor-Ligand Interactions. J. Vis. Exp. (Pending Publication), e21453, doi: (2023).

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