A Biocompatible “Split Luciferin” Reaction and Its Application for Non‐Invasive Bioluminescent Imaging of Protease Activity in Living Animals

Aurélien Godinat1, Ghyslain Budin1, Alma R. Morales1, Hyo Min Park2, Laura E. Sanman3, Matthew Bogyo4, Allen Yu2, Andreas Stahl2, Elena A. Dubikovskaya1

1 Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology of Lausanne, Lausanne, 2 Department of Nutritional Science and Toxicology, University of California Berkeley, Berkeley, California, 3 Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California, 4 Department of Pathology, Stanford University School of Medicine, Stanford, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140047
Online Posting Date:  September, 2014
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The great complexity of many human pathologies, such as cancer, diabetes, and neurodegenerative diseases, requires new tools for studies of biological processes on the whole organism level. The discovery of novel biocompatible reactions has tremendously advanced our understanding of basic biology; however, no efficient tools exist for real‐time non‐invasive imaging of many human proteases that play very important roles in multiple human disorders. We recently reported that the “split luciferin” biocompatible reaction represents a valuable tool for evaluation of protease activity directly in living animals using bioluminescence imaging (BLI). Since BLI is the most sensitive in vivo imaging modality known to date, this method can be widely applied for the evaluation of the activity of multiple proteases, as well as identification of their new peptide‐specific substrates. In this unit, we describe several applications of this “split luciferin” reaction for quantification of protease activities in test tube assays and living animals. Curr. Protoc. Chem. Biol. 6:169‐189 © 2014 by John Wiley & Sons, Inc.

Keywords: protease activity; bioocompatible reaction; non‐invasive; bioluminescent imaging; in vivo

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Table of Contents

  • Introduction
  • Basic Protocol 1: In Vitro (Cell‐Free) Bioluminescence Imaging of Protease Activity Using “Split Luciferin” Reaction
  • Alternate Protocol 1: In Vitro (Cell‐Free) Imaging of Thrombin Protease Activity
  • Basic Protocol 2: Real‐Time Non‐Invasive Imaging of Caspase‐3/7 Activities in Transgenic Reporter Mice (FVB‐Luc+)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: In Vitro (Cell‐Free) Bioluminescence Imaging of Protease Activity Using “Split Luciferin” Reaction

  • Caspase buffer (see recipe)
  • 200 nM of purified caspase‐3 solution (see recipe)
  • 800 μM D‐cysteine solution (see recipe)
  • 800 μM DEVD‐(D‐Cys) peptide solution (see recipe)
  • 400 μM 6‐amino‐2‐cyanobenzothiazole (NH 2‐CBT; see recipe)
  • 60 μg/ml Firefly luciferase enzyme solution (see recipe)
  • V‐shaped 96‐well plate (Vitaris, cat. no. 3897‐COR)
  • 37°C incubator
  • Black 96‐well plate (BD Falcon, cat. no. 353219)
  • Bioluminescence plate reader or imager (Spectramax M5, Molecular Devices)

Alternate Protocol 1: In Vitro (Cell‐Free) Imaging of Thrombin Protease Activity

  • Thrombin stock solution (see recipe)
  • Thrombin buffer (see recipe)
  • D‐cysteine solution (see recipe)
  • 500 μM GGR‐(D‐Cys) peptide solutions (see recipe)
  • 500 μM NH 2‐CBT solution (see recipe)
  • 60 μg/ml Firefly luciferase enzyme solution (see recipe)
  • V‐shaped 96‐well plate (Vitaris, cat. no. 3897‐COR)
  • 37°C incubator
  • Black 96‐well plate (BD Falcon, cat. no. 353219)
  • Bioluminescence plate reader (Spectramax Gemini, Molecular Devices) or camera‐like IVIS Spectrum (Perkin Elmer)

Basic Protocol 2: Real‐Time Non‐Invasive Imaging of Caspase‐3/7 Activities in Transgenic Reporter Mice (FVB‐Luc+)

  • 3.1 mg/ml D‐luciferin potassium salt (Intrace Medical) solution (see recipe)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 20012‐068), sterile
  • FVB‐Luc+ mice [FVB‐Tg(CAG‐luc,‐GFP)L2G85Chco/J, The Jackson Laboratory; Cao et al., ]
  • Isoflurane for anesthesia (Phoenix)
  • 50 μg/ml lipopolysaccharide (LPS) (from Salmonella typhosa, Sigma‐Aldrich, cat. no. L7895) solution (see recipe)
  • 133.5 mg/ml D‐(+)‐galactosamine hydrochloride (D‐GalN) solution (see recipe)
  • 5.65 mg/ml DEVD‐(D‐Cys) peptide solution (see recipe)
  • 8.5 mg/ml 6‐amino‐2‐cyanobenzothiazole (NH 2‐CBT) solution (see recipe)
  • Dimethyl sulfoxide (DMSO), sterile
  • 0.5‐ml insulin syringes (U‐100 insulin syringe, 28‐G × ½‐in. needle; BD, cat. no. 329465)
  • Clear, Plexiglas box
  • Bioluminescence imaging system for small laboratory animals (IVIS Spectrum, PerkinElmer) or similar device
  • 37°C heating pad
NOTE: The following procedure is described for an average 25 g FVB‐luc+ mouse. If the mice used have different weight, it is advised to adjust the concentrations of solutions in order to keep the doses constant.
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