Protocol for generation of transmitochondrial cybrids under pyruvate/uridine-supplemented conditions using a microfluidic device
Ken-Ichi Wada, Kazuo Hosokawa, Yoshihiro Ito, Mizuo Maeda, Yui Harada, Yoshikazu Yonemitsu
TL;DR
This paper introduces a new protocol using a microfluidic device to generate transmitochondrial cybrids even when pyruvate and uridine are present in the medium.
Contribution
A novel microfluidic-based method for generating transmitochondrial cybrids under PU-supplemented conditions.
Findings
The microfluidic device enables mitochondrial transfer between single cells.
mtDNA repopulation and typing in cybrids can be confirmed using RFLP analysis.
The protocol works effectively even with pyruvate/uridine in the medium.
Abstract
Under conventional approaches, selective culture with a pyruvate/uridine (PU)-free medium is essential for generating transmitochondrial cybrids. Here, we present a protocol for generating transmitochondrial cybrids using a microfluidic device, which works even under PU-supplemented conditions. We describe steps for preparation of the microfluidic device, partial cell fusion (mitochondrial transfer), and harvest of transmitochondrial cybrids. We then detail procedures for confirmation of mtDNA repopulation in cybrids and mtDNA typing by restriction fragment length polymorphism (RFLP) analysis. For complete details on the use and execution of this protocol, please refer to Wada et al.1 •Protocol for the generation of transmitochondrial cybrids using a microfluidic device•Instructions for the fabrication of a microfluidic device•Steps for mitochondrial transfer between single cells…
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Taxonomy
TopicsInnovative Microfluidic and Catalytic Techniques Innovation