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…

Divulgaciones

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

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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).

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