Antibody Conjugated Nanoparticle-based Quantification Using Dark Field Microscopy: A Procedure to Capture and Quantify Specific Exosomes from Body Fluids

Published: April 30, 2023

Abstract

Source: Wan, M. et al. Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes. J. Vis. Exp. (2019)

This video describes the use of low-magnification, dark-field microscopy to quantify specific exosomes in small volume biofluidic samples. The exosomes thus identified can serve as potential cancer biomarkers.

Protocol

1. Preparation of Nanoparticle Probes NOTE: This assay utilizes Functionalized Gold Nanorods (AuNRs; 25 nm diameter x 71 nm length) that are covalently conjugated with neutravidin polymers (AV) and have a surface plasmon resonance peak that produces a red (641 nm peak) scattering signal upon DFM illumination. Wash 40 µL of AuNR-AV (2.56 x 1011 particles) three times with 200 µL PBS (pH 7.0) by centrifugation and aspiration…

Divulgaciones

The authors have nothing to disclose.

Materials

Eppendorf Repeater stream Fisher Scientific 05-401-040
Eppendorf Research plus Eppendorf 3120000011 0.1 – 2.5 µL, dark gray
Functionalized Gold Nanorods Nanopartz C12-25-650-TN-DIH-50-1 In vitro neutravidin polymer
functionalization
HulaMixer Sample Mixer Thermo Fisher Scientific 15920D
Incu-shaker 10L Benchmark Scientific H1010
Inverted Research Microscope Nikon Ti-DH With Dark field condenser, DS-Ri2
camera, and Ti-SH-U universal
holder, and motorized stage
NIS-Elements Nikon Microscope imaging software
Phosphate Buffered Saline (1X) GE Healthcare Life Sciences SH30256.02 HyClone
Protein A/G Treated Glass
Substrate Slides
Arrayit Corp. AGMSM192BC Premium microarray substrate
Q500 Sonicator Qsonica, LLC Q500-110 With standard probe (#4220)
Superblock blocking buffer Thermo Scientific
TWEEN 20 Sigma Life Sciences 9005-64-5

Tags

Play Video

Citar este artículo
Antibody Conjugated Nanoparticle-based Quantification Using Dark Field Microscopy: A Procedure to Capture and Quantify Specific Exosomes from Body Fluids. J. Vis. Exp. (Pending Publication), e20498, doi: (2023).

View Video