This video describes a co-culture assay of glioblastoma cells on micropatterned neurons, which mimic the myelinated neuronal tracts invasion by the glioblastoma stem-like cells as they migrate on neurons in the peritumoral spaces. This assay can help analyze the effects of pharmacological drugs that inhibit glioblastoma cells' migration on neurons in a glioma.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of the Patterned Slides Substrate Preparation for Micropatterning Treat 18 mm circular glass coverslips by air/plasma activation for 5 min. Place the coverslips in a closed chamber with 100 μL of (3-aminopropyl) triethoxysilane in a desiccator for 1 h. Incubate wi…
Declarações
The authors have nothing to disclose.
Materials
(3-aminopropyl) triethoxysilane
Sigma
440140-100ML
The amino group is useful for the bioconjugation of mPEG-SVA
96-well round-bottom plate
Sarstedt
2582624
Used to prepare spheroids
Accutase
Gibco
A11105-01
Stored at -20 °C (long-term) or 4°C (short-term), sphere dissociation enzyme
B27
Gibco
12587
Stored at -20 °C, defrost before use
Basic Fibroblast Growth Factor
Peprotech
100-18B
Stored at -20 °C, defrost before use
Countess Cell Counting Chamber Slides
Invitrogen
C10283
Used to cell counting
Coverslips
Marienfeld
111580
Cell culture substrate
Dessicator cartridges
Sigma
Z363456-6EA
Used to reduce moisture during (3-aminopropyl) triethoxysilane treatment
DPBS 10x
Pan Biotech
P04-53-500
Stored at 4 °C
Flask 75 cm²
Falcon
10497302
HBSS
Sigma
H8264-500ML
Heparin sodium
Sigma
H3149-100KU
Stored at 4 °C
Laminin
114956-81-9
Promotes neuronal adhesion
Flask 75 cm²
Falcon
10497302
Heparin sodium
Sigma
H3149-100KU
Stored at 4 °C
Leonardo software
Loading of envisioned micropatterns
MetaMorph Software
Molecular Devices LLC
NA
Microscopy automation software
Methylcellulose
Sigma
M0512
Diluted in NBM for a 2% final concentration
Neurobasal medium
Gibco
21103-049
Stored at 4 °C
Nikon TiE (S Fluor, 20x/0.75 NA)
Inverted microscope equipped with a motorized stage
Co-Culturing Glioblastoma Cells on Patterned Neurons: A Technique to Monitor the Migration of Glioblastoma Cells on Rat Hippocampal Neurons In Vitro. J. Vis. Exp. (Pending Publication), e20680, doi: (2023).