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.
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.
2. Glass Bead Silanization
3. Bead Functionalization
4. Cell Preparation
NOTE: To purify the cells, follow standard cell purification protocols corresponding to the type of cells in use, for example, T-cells or certain cell lines.
5. Preparation for Micropipettes and a Cell Chamber
4. BFP experiment
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 adapts an experiment chamber and three micropipette manipulation systems. (E and F) Micrographs of BFP setting in an experimental chamber. (E) Micropipette assembly showing the probe pipette (left), target pipette (upper right), and helper pipette (lower right). (F) Probe bead placement. A probe bead was manipulated by a helper pipette and attached to an RBC apex to form a force probe.
Figure 2: BFP scheme and its test cycle.
(A) Video micrograph depicting a force probe (left) and a target T-cell (right) aspirated by their respective pipettes. The stationary force probe consists of a swollen RBC and an attached ligand-bearing bead. The receptor-bearing T-cell (target) is mounted to a piezo translator aligned opposite to the probe. The ROI is indicated in green. The edge tracker is indicated in a blue line. The insert depicts the ligand (pMHC, bead side) and receptor (TCR, T-cell side) pair on the two opposing surfaces in the area marked in orange. (B) The intensity profile of the bead edge in (A). The ROI region in the x-direction is plotted as the x-axis (in pixel number) and the light intensity (in grayscale value) averaged by binning 30 pixels along the y-direction. (C) The deflection of the RBC and the position of the bead and the target (T-cell) in a test cycle of force clamp assay. The vertical and horizontal dashed lines indicate the zero-force position of the RBC apex and the time course, respectively. The line edge tracker of the RBC deformation is shown in blue on each panel. The same yet less steps are adopted in adhesion frequency assay (which lacks the steps of “clamp” and “dissociate”) and thermal fluctuation assay (which lacks the step of “dissociate”).
The authors have nothing to disclose.
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 |