Click-iT ® Plus OPP Alexa Fluor ® Protein Synthesis Assay in Embryonic Cells

Yan Li; Xu Ji; Lu Chang; Jianan Tang; Min-Min Hua; Jing Liu; Christopher O'Neill; Xuefeng Huang; Xingliang Jin

doi:10.21769/BioProtoc.4441

Click-iT ^® Plus OPP Alexa Fluor ^® Protein Synthesis Assay in Embryonic Cells

Bio Protoc. 2022 Jun 5;12(11):e4441. doi: 10.21769/BioProtoc.4441.

Authors

Yan Li¹, Xu Ji², Lu Chang², Jianan Tang³, Min-Min Hua³, Jing Liu², Christopher O'Neill⁴, Xuefeng Huang¹, Xingliang Jin^{1

4}

Affiliations

¹ Reproductive Medicine Center, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, Zhejiang Province, China.
² School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 211816, Jiangsu Province, China.
³ NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Fudan University, 200032, Shanghai, China.
⁴ Sydney Center for Regenerative and Developmental Medicine, Kolling Institute for Medical Research, Sydney Medical School, University of Sydney, St. Leonards, New South Wales, 2065, Australia.

Abstract

This protocol describes a method to assess relative changes in the level of global protein synthesis in the preimplantation embryo using the Click-iT ^® Plus OPP Protein Synthesis Assays. In this assay, O-propargyl-puromycin (OPP), an analog of puromycin, is efficiently incorporated into the nascent polypeptide of newly translated proteins in embryonic cells. OPP is fluorescently labeled with a photostable Alexa Fluor ^TM dye and detected with fluorescence microscopy. The intensity of the fluorescence is quantitatively analyzed. This is a fast, sensitive, and non-radioactive method for the detection of protein synthesis in early embryo development. It provides a tool for analyzing the temporal regulation of protein synthesis, as well as the effects of changes in the embryonic microenvironment, and pharmacological and genetic modulations of embryo development. Graphical abstract: Figure 1. Brief overview of the procedures of the Click-iT ^® Plus OPP Alexa Fluor ^® protein synthesis assay in embryonic cells. (A) Set up OPP treatments: (1) Set up microdrops containing 50 µL of OPP working solution and label different treatments on the back of culture dishes ( e.g. , T0, T1, T2, and T3); (2) The drops are overlain with 2-3 mm heavy paraffin oil and then equilibrated in incubator for 2 h; (3) Collect the embryos from female reproductive tracts or following in vitro culture in desired treatments; (4) Culture embryos in the equilibrated OPP working solution for 2-6 h. ( B ) Example of OPP detection procedures working with 60-well plates labeled as T0, T1, T2, T3, T4, and T5 for different treatments: (1) The first 60-well plate is used for the procedures of washing, fixation, permeabilization, and Click-iT ^® OPP detection. (2) The second 60-well plate is for DNA staining and washing. (C) Slide preparation: (1) Label the required number of slides and set up vaseline coverslip supports; (2) Add mounting medium; (3) Transfer embryos into mounting medium; (4) Set coverslip; (5) Seal the coverslip with nail polish.

Keywords: Embryo development; Maternal-embryonic transition; O-propargyl-puromycin; Protein synthesis.