Matrix-Based Tumor Invasion Model Using Microglia Glioblastoma Co-Culture

Abstract

Source: Coniglio, S., et al. Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion. J. Vis. Exp. (2016)

In this video, glioblastoma and microglia cells are mixed with a polymer matrix solution and transferred to a porous chamber insert where a gel forms, embedding the cells. Microglia stimulate the migration of glioblastoma cells through the porous insert, mimicking tumor cell invasion into surrounding tissue, Cells are then fixed and prepared for further analysis.

Protocol

1. Fluorescent Labeling of Cells

NOTE: Label glioblastoma cell lines and microglia with fluorescent dyes 9. Alternatively, generate cell lines that constitutively express fluorescent proteins such as GFP/RFP.

1. Plate cells on a 6 well plate such that they will be 70 - 80% confluent on the day of staining. For the murine glioblastoma cell line GL261 and human glioblastoma cell line U87, plate 1 x 10⁶ and 1.5 x 10⁶ cells, respectively, on a 6 cm dish 24 hr before staining.
2. Prepare fluorescent cell stain dye solution in DMSO (Dimethyl sulfoxide) and add 5 µM dye t....

Results

Figure 1: Schematic of Two Different Protocols for Assaying Microglia/Macrophage-stimulated Glioblastoma Cell Invasion. Top panel illustrates pre-coated matrix chamber invasion assay. Bottom panel illustrates 3D matrix invasion assay.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Corning BioCoat Matrigel Invasion Chamber: With BD Matrigel Matrix	Corning/Fisher Scientific	Cat: 354481
Macrophage Serum Free Media (MSFM) (500 ml)	Life Technologies	12065-074
CellTracker Red CMTPX Dye	Life Technologies/Molecular Probes	C34552
CellTracker Green CMFDA Dye	Life Technologies/Molecular Probes	C2925
GL261 cell line	National Cancer Institute (NCI)
THP-1 cell line	American Tissue Type Culture Collection	ATCC TIB-202
RPMI 1640 Medium (500 ml)	Life Technologies/Gibco	11875-093
Formaldehyde solution	Sigma Aldrich	F1635
Corning Transwell polycarbonate membrane cell culture inserts (8 µM pore) 48 per pack.	Corning	CLS3422
Cultrex 3-D Culture Matrix Reduced Growth Factor Basement Membrane Extract, PathClear	Trevigen	3445-005-01
Fetal calf serum (FBS)	Life Technologies	Cat: 10500064
Bovine Serum Albumin, Fraction V, Heat Shock Treated	Fisherscientific	BP1600-100
0.5M EDTA	ThermoFisher Scientific	15575-020