Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Yunfeng Chen; Baoyu Liu; Lining Ju; Jinsung Hong; Qinghua Ji; Wei Chen; Cheng Zhu

doi:10.3791/52975

Fluorescence Biomembrane la Force Probe: quantification simultanée de Kinetics récepteur-ligand e...

JoVE Journal
Bioengineering

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Bioengineering

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Fluorescence Biomembrane la Force Probe: quantification simultanée de Kinetics récepteur-ligand et induits Reliure-signalisation intracellulaire sur une cellule unique

Full Text

13,051 Views

14:09 min

August 4, 2015

DOI: 10.3791/52975-v

Yunfeng Chen*¹, Baoyu Liu*², Lining Ju*³, Jinsung Hong*¹, Qinghua Ji^4,5, Wei Chen⁶, Cheng Zhu¹

¹Woodruff School of Mechanical Engineering, Petit Institute for Bioengineering and Biosciences,Georgia Institute of Technology, ²Coulter Department of Biomedical Engineering,Georgia Institute of Technology, ³Charles Perkins Centre,The University of Sydney, ⁴Institute of Biophysics, Laboratory of RNA Biology,Chinese Academy of Sciences, ⁵University of Chinese Academy of Sciences, ⁶School of Medicine and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,Zhejiang University

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This article describes a technique for concurrently measuring receptor-ligand binding kinetics while observing calcium signaling in a single T lymphocyte. The method utilizes biotinylated red blood cells as force transducers and microscale glass beads for functionalization.

Key Study Components

Area of Science

Cell biology
Immunology
Biophysics

Background

Understanding receptor-ligand interactions is crucial for immunological research.
Calcium signaling plays a key role in T cell activation.
Simultaneous measurement techniques enhance data accuracy.
Force transduction methods can provide insights into binding kinetics.

Purpose of Study

To measure receptor-ligand binding kinetics in real-time.
To observe calcium signaling induced by binding events.
To develop a method applicable to single-cell analysis.

Methods Used

Isolation of biotin and adjustment of osmolarity of human red blood cells.
Functionalization of microscale glass beads with ligands.
Isolation and incubation of T cells with calcium dye.
Preparation of micro pipettes in an experimental chamber.

Main Results

Successful measurement of receptor-ligand binding kinetics.
Real-time monitoring of intracellular calcium levels.
Demonstration of the fluorescence bio membrane force probe technique.
Insights into T cell adhesion dynamics.

Conclusions

The technique provides a powerful tool for studying single-cell interactions.
It enhances understanding of T cell activation mechanisms.
Future applications may extend to other cell types and signaling pathways.

Frequently Asked Questions

What is the significance of measuring receptor-ligand binding kinetics?

Measuring these kinetics helps understand the strength and duration of interactions critical for T cell activation.

How does calcium signaling relate to T cell function?

Calcium signaling is essential for various T cell functions, including activation, proliferation, and cytokine production.

What are the advantages of using single-cell techniques?

Single-cell techniques allow for the observation of heterogeneity in cellular responses that bulk measurements may miss.

What role do biotinylated red blood cells play in this method?

They serve as ultra-sensitive force transducers to measure binding interactions with high precision.

Can this method be applied to other cell types?

Yes, while this study focuses on T lymphocytes, the technique can be adapted for other cell types.

What is the importance of using calcium dyes in this experiment?

Calcium dyes enable real-time visualization of intracellular calcium levels, providing insights into signaling dynamics.

Nous décrivons une technique permettant de mesurer simultanément la cinétique de liaison du récepteur unique et du ligand régulée par la force et l’imagerie en temps réel de la signalisation calcique dans un seul lymphocyte T.

L’objectif global de cette procédure est de mesurer simultanément le type de récepteur et la liaison et d’observer la signalisation calcique induite par la liaison sur une seule cellule. Ceci est accompli en isolant d’abord la biotine et en ajustant l’osmolarité des globules rouges humains ou GR pour les utiliser comme un transducteur de force ultra-sensible. La deuxième étape consiste à fonctionnaliser des billes de verre à l’échelle microscopique, ce qui comprend le nettoyage, la dilatation et l’enrobage de ligands.

Ensuite, les lymphocytes T sont isolés et incubés avec le colorant calcique moins de 2h00 du matin, ce qui permet d’observer la concentration intracellulaire des ions calcium. La dernière étape consiste à préparer les micro-pipettes dans la chambre expérimentale qui sont spécialement nécessaires à la configuration de l’expérience. En fin de compte, la sonde de force biomembranaire de fluorescence ou technique BFP est utilisée pour montrer l’adhésion d’une cellule à une bille recouverte d’un ligand, mesurer la cinétique de liaison du ligand du récepteur et, pendant ce temps, surveiller le niveau de calcium intracellulaire.

View the full transcript and gain access to thousands of scientific videos

Explore More Videos

Bioengineering Numéro 102 cellule unique seule molécule liaison récepteur-ligand cinétique la spectroscopie de fluorescence et de la force l'adhérence mécano-transduction le calcium