Flow cytometric analysis of phosphatidylcholine metabolism using organelle-selective click labeling

Masaki Tsuchiya; Nobuhiko Tachibana; Itaru Hamachi

doi:10.1016/j.xpro.2023.102525

Flow cytometric analysis of phosphatidylcholine metabolism using organelle-selective click labeling

STAR Protoc. 2023 Sep 15;4(3):102525. doi: 10.1016/j.xpro.2023.102525. Epub 2023 Aug 26.

Authors

Masaki Tsuchiya¹, Nobuhiko Tachibana², Itaru Hamachi³

Affiliations

¹ Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan; PRESTO (Precursory Research for Embryonic Science and Technology, JST), Sanbancho, Chiyodaku, Tokyo 102-0075, Japan. Electronic address: tsuchiya.masaki.7a@kyoto-u.ac.jp.
² Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan; PRESTO (Precursory Research for Embryonic Science and Technology, JST), Sanbancho, Chiyodaku, Tokyo 102-0075, Japan.
³ Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan; ERATO (Exploratory Research for Advanced Technology, JST), Sanbancho, Chiyodaku, Tokyo 102-0075, Japan. Electronic address: ihamachi@sbchem.kyoto-u.ac.jp.

Abstract

Here, we present a protocol to analyze phosphatidylcholine (PC) metabolism in mammalian cells using organelle-selective click labeling coupled with flow cytometry (O-ClickFC). We describe steps for the metabolic incorporation of azide-choline into PC. We then detail fluorescent labeling of the azide-modified PC with organelle-targeting clickable dyes in the ER-Golgi, plasma membrane, and mitochondria, and by flow cytometry. This protocol is optimized for flow cytometric quantification of the labeled PC at the organelle level within single live cells. For complete details on the use and execution of this protocol, please refer to Tsuchiya et al. (2023).¹.

Keywords: Chemistry; Flow Cytometry/Mass Cytometry; Metabolism; Molecular/Chemical Probes.

MeSH terms

Animals
Azides*
Flow Cytometry / methods
Golgi Apparatus*
Mammals
Mitochondria
Phosphatidylcholines

Substances

Azides
Phosphatidylcholines