Fluorescent Labeling of Glioma Cells: A Lentiviral Vector-based Transfection Method to Obtain Glioma Cells Expressing Fluorescent Protein for Deep Tissue Imaging

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

1. Produce and purify lentivirus according to the protocol described below:
   1. Observe the dishes seeded on Day 1. When cells are 80–90% confluent they are ready for transfection.
      ​NOTE: The cells should still have room to undergo 1–2 cell divisions
   2. Prepare the plasmid mix by aliquoting the four plasmids into a 50 mL tube. For a 12 × 15 cm dis....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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