Fluorescent Labeling of Glioma Cells: A Lentiviral Vector-based Transfection Method to Obtain Glioma Cells Expressing Fluorescent Protein for Deep Tissue Imaging
Published: April 30, 2023
Abstract
Source: Benitez, J. A. et al. Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts. J. Vis. Exp. (2018)
In this video, we describe a protocol for genetically engineering glioma cells to express infrared fluorescent protein or iRFP. These fluorescent-tagged glioma cells hold utility in deep tissue imaging to understand the development of brain tumors.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Labeling of Glioblastoma Cells with FP635 or FP720 Construct Produce and purify lentivirus according to the protocol described below: Observe the dishes seeded on Day 1. When cells are 80–90% confluent they are ready for transfection. NOTE: The cells should still have ro…
Divulgazioni
The authors have nothing to disclose.
Materials
| DMEM/High Glucose | HyClone/GE | SH30022.1 | |
| DMEM/F12 1:1 | Gibco | 11320-082 | |
| FBS | HyClone/GE | SH30071.03 | |
| Accutase | Innovative cell technologies | AT-104 | |
| Trypsin | HyClone/GE | SH30236.01 | |
| B27 supplement | Gibco | 17504044 | |
| Human recombinant EGF | Stemcell Technologies | 2633 | |
| Human recombinant FGF | Stemcell Technologies | 2634 | |
| DPBS | Corning | 21-031-00 | |
| FACS tubes | Falcon | 352235 | |
| DAPI | ThermoFisher Scientific | 62248 | |
| p24 ELISA | Clontech | 632200 | |
| Cell Sorter | Sony | SH8007 | |
| Ultra-centrifuge Optima L-80 XP | Beckman Coulter | 392049 | |
| Tissue Culture 100mm Dishes | Olympus Plastics | 25-202 | |
| Tissue Culture 150mm Dishes | Olympus Plastics | 25-203 | |
| Tissue Culture Flasks T75 | Corning | 430720U | |
| 50 mL conical tubes | Corning | 430290 | |
| Plasmids: lentiviral transfer vector | Verma Lab Salk Institute for Biological Studies | ||
| Lentiviral packaging vectors | pMDL pRev and pVSVG | K4975-00 | |
| Endotoxin free maxipreps | Endo-free Maxiprep Kit Qiagen | 12632 | |
| 1X— HBSS | Invitrogen | 14025-092 | |
| 15 cm tissue culture dishes | |||
| 0.45 µm filter units 500 mL capacity | |||
| Conical bottom ultracentrifugation tubes | Beckman | 358126 | |
| Round bottom ultracentrifugation tubes | Beckman | 326819 | |
| 24-well tissue culture plates | |||
| Fluorescent microscope | |||
| Multichannel pipettor | |||
| ELISA microplate reader |
Tags
Citazione di questo articolo
Fluorescent Labeling of Glioma Cells: A Lentiviral Vector-based Transfection Method to Obtain Glioma Cells Expressing Fluorescent Protein for Deep Tissue Imaging. J. Vis. Exp. (Pending Publication), e20676, doi: (2023).