Using DNA-Based Tension Probes to Assess Receptor Forces Applied by Immune Cells

Published: February 29, 2024

Abstract

Source: Ma, R. et al., DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells. J. Vis. Exp. (2021)

The video demonstrates a method for assessing receptor forces applied by immune cells using polyethylene glycol-anchored gold nanoparticles and DNA hairpin tension probes. CD8-positive T cell interactions trigger probe unfolding, emitting fluorescence while locking strands sustain the signal for quantifying the forces applied by immune cells.

Protocol

1. Oligonucleotide preparation Dissolve the ligand strand deoxyribonucleic acid (DNA) in water (18.2 MΩ resistivity, used throughout the whole protocol). Vortex and spin down the solution with a tabletop centrifuge. Tune the volume of water such that the final concentration is 1 mM. Validate the concentration by using a nanodrop spectrophotometer to measure the absorbance at 260 nm and determine the final concentration based on the extinction coefficient of the oligonucleotide. <br …

Representative Results

Figure 1: Examples of oligonucleotide preparation. (A) HPLC chromatogram of Cy3B ligand strand purification; (B) MALDI-TOF-MS spectra of the product; (C) Table of the calculated mass and found m/z peaks of the starting material and the labeled oligonucleotide.  <img alt="Figur…

Açıklamalar

The authors have nothing to disclose.

Materials

 3-hydroxypicolinic acid (3-HPA) Sigma 56197 Maldi-TOF-MS matrix
 mPEG-SC Biochempeg MF001023-2K Surface prep
(3-Aminopropyl)triethoxysilane Acros AC430941000 Surface prep
10x Red blood cell lysis buffer Biolegend 00-4333-57 Buffer
8.8 nm gold nanoparticles, tannic acid Nanocomposix customized order Surface prep
Atto647N NHS ester Sigma 18373-1MG-F Fluorophore, oligo prep
Attofluor Cell Chamber, for microscopy Thermo Fisher Scientific A7816 Imaging
BD Syringes only with Luer-Lok BD bioscience 309657 Cells
Biotinylated anti-mouse CD3e ebioscience 13-0031-82 Antibody/ligand
Biotinylated pMHC ovalbumin (SIINFEKL) NIH Tetramer Core Facility at Emory University NA Antibody/ligand
Bovine serum albumin Sigma 735078001 Block non-specific interactions
Cell strainers Biologix 15-1100 Cells
Coverslip Mini-Rack, teflon Thermo Fisher Scientific C14784 Surface prep
Cy3B NHS ester GE Healthcare PA63101 Fluorophore, oligo prep
Dulbecco's phosphate-buffered saline (DPBS) Corning 21-031-CM Buffer
Ethanol Sigma 459836 Surface prep
Hank's balanced salts (HBSS) Sigma H8264 Buffer
Hydrogen peroxide Sigma H1009 Surface prep
LA-PEG-SC Biochempeg HE039023-3.4K Surface prep
Midi MACS (LS) startup kit Miltenyi Biotec 130-042-301 Cells
Mouse CD8+ T cell isolation kit Miltenyi Biotec 130-104-075 Cells
Nanosep MF centrifugal devices Pall laboratory ODM02C35 Oligo prep
No. 2 round glass coverslips VWR 48382-085 Surface prep
NTA-SAM Dojindo Molecular Technologies N475-10 Surface prep
P2 gel Bio-rad 1504118 Oligo prep
Sulfuric acid EMD Millipore Corporation SX1244-6 Surface prep
Sulfo-NHS acetate Thermo Fisher Scientific 26777 Surface prep
Equipment
Agilent AdvanceBio Oligonucleotide C18 column, 4.6 x 150 mm, 2.7 μm 653950-702 Oligonucleotide preparation
Barnstead Nanopure water purifying system Thermo Fisher Water
CFI Apo 100× NA 1.49 objective Nikon Microscopy
Cy5 cube CHROMA Microscopy
Evolve electron multiplying charge coupled device (EMCCD) Photometrics Microscopy
High-performance liquid chromatography Agilent 1100 Oligonucleotide preparation
Intensilight epifluorescence source Nikon Microscopy
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) Voyager STR Oligonucleotide preparation
Nanodrop 2000 UV-Vis Spectrophotometer Thermo Fisher Oligonucleotide preparation
Nikon Eclipse Ti inverted microscope Nikon Microscopy
Nikon Perfect Focus System Nikon Microscopy
NIS Elements software Nikon Microscopy
Quad band TIRF 405/488/561/647 cube CHROMA Microscopy
RICM cube CHROMA Microscopy
TIRF launcher with 488 nm (50 mW), 561 nm (50 mW), and 640 nm Coherent Microscopy
TRITC cube CHROMA Microscopy
Oligo name 5' modification / 3' modification Sequence (5' to 3') Use
15mer amine locking strand 5' modification: no modification
3' modification: /3AmMO/
AAA AAA CAT TTA TAC CCT ACC TA Locking real-time tension signal
15mer Atto647N locking strand 5' modification: Atto647N
3' modification: /3AmMO/
AAA AAA CAT TTA TAC CCT ACC TA Locking real-time tension signal
15mer non-fluoresccent locking strand 5' modification: no modification
3' modification: no modification
A AAA AAC ATT TAT AC Locking real-time tension signal for quantitative analysis
4.7 pN hairpin strand 5' modification: no modification
3' modification: no modification
GTGAAATACCGCACAGATGCGT
TTGTATAAATGTTTTTTTCATTTAT
ACTTTAAGAGCGCCACGTAGCC
CAGC
Hairpin probe
Amine ligand strand 5' modification: /5AmMC6/
3' modification: /3Bio/
CGCATCTGTGCG GTA TTT CAC TTT Hairpin probe
BHQ2 anchor strand 5' modification: /5ThiolMC6-D/
3' modification: /3BHQ_2/
TTTGCTGGGCTACGTGGCGCTCTT    Hairpin probe
Cy3B ligand strand 5' modification: Cy3B
3' modification: /3Bio/
CGCATCTGTGCG GTA TTT CAC TTT Hairpin probe
Unlocking strand 5' modification: no modification
3' modification: no modification
TAG GTA GGG TAT AAA TGT TTT TTT C Unlocking accumulated tension signal

Etiketler

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Bu Makaleden Alıntı Yapın
Using DNA-Based Tension Probes to Assess Receptor Forces Applied by Immune Cells. J. Vis. Exp. (Pending Publication), e21989, doi: (2024).

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