In Vivo Bioluminescence Imaging of Human Neural Progenitor Cells in a Mouse Brain

Abstract

Source: Weber, R. Z., et al., Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain. J. Vis. Exp. (2022)

This video demonstrates the method for in vivo bioluminescence imaging of genetically modified human neural progenitor cells injected in a mouse brain. The process involves luciferin injection, imaging preparation, and detection of bioluminescent signals from the transplanted cells at the injection site.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

In vivo imaging

1. Preparation of luciferin
   1. Thaw D-luciferin potassium salt at room temperature (RT) and prepare a fresh stock solution of D-luciferin at 30 mg/mL in PBS.
   2. Sterilize the stock solution through a 0.22 µm syringe filter.
      NOTE: Immediate use of the working solution is recommended. If necessary, dissolved luciferin can be stored at -20 °C. However, prolonged storage may result in the degradation of the signal. Luciferin is a light-sensitive reagent; keep it out of direct light whenever possible. Alternative substrates may also be considered, e.g., cycluc, to improve the resolution limit.
2. Imaging
   1. Initial setup
      NOTE: Bioluminescence imaging was performed using an in vivo imaging system (see the Table of Materials) consisting of a dark chamber and a cooled charge-coupled device (CCD) camera.
      1. Double-click the Living Image software icon and select a user ID from the drop-down list.
      2. Click Initialize in the control panel that appears. Once the initialization process is completed, the temperature box in the control panel will turn green.
      3. In the control panel, check the Luminescent and Photograph boxes and select Auto exposure (~60 s). Select a field of view (D/12.5cm was chosen for this protocol). Enter the subject height (1.5 cm) and select the use subject height focus option. Manually set the following parameters: large binning, f/2, blocked excitation filter, and open emission filter.
   2. Determine the injection amount of D-luciferin at 300 mg/kg body weight.
      NOTE: The standard recommended dose is 150 mg/kg of D-luciferin. This procedure was adjusted according to a protocol reporting higher sensitivity using 300 mg/kg.
   3. Inject the luciferin intraperitoneally (i.p.).​
      NOTE: If the animal needs to be sedated before injection, be aware that it may extend the peak luciferase expression time.
   4. Wait for 5 min, then anesthetize animals with a continuous supply of isoflurane (4.5% in the induction phase and 1.5-2% in the maintenance phase during the imaging procedure).
   5. Apply ophthalmic lubricant to the eyes, then shave the sedated animals on the head region using a conventional hair shaver. Place the animals in the imaging chamber and start imaging 15 min after the luciferin injection by clicking Acquire in the control panel.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Consumables
Syringe filter; 0.22 µm	TPP	99722
Equipment
Heating pad			Product of choice
In-Vivo imaging system (IVIS Lumina III with Living Imaging 4.2 software package)	Perkin Elmer	CLS136334
Isoflurane vaporizer	Provet AG	330724
Pharmaceuticals and Reagents
D-Luciferin Potassium Salt	Perkin Elmer	122799
Ethanol; 70%			Product of choice
Isoflurane (Isofluran (1-Chlor-2,2,2-trifluorethyl-difluoromethyl­ether) 99.9%)	Provet AG	All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland
Ophtalmic lubricant (Retinol palmitat: 15,000 UI)	Bausch & Lomb Swiss AG	All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland