Fluorescence Based Transwell Macrophage Attraction Assay: An In Vitro Method to Study Tumor-Macrophage Interaction Through Chemical-Induced Chemotaxis

Abstract

Source: An, Z. et al. In Vitro Assay to Study Tumor-macrophage Interaction. J. Vis. Exp. (2019)

In this video, we study tumor-macrophage interaction using fluorescence-based transwell macrophage interaction assay. The assay involves the analysis of the migration of macrophages through chemical-induced chemotaxis.

Protocol

1. In Vitro Macrophage Attraction Assay

1. Preparation of Conditioned Media

1. Warm up the tumor cell maintenance medium at a 37 °C water bath for 20 min.
2. Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels. Take the cell detachment solution from the fridge. Spray the bottles of the medium and the cell detachment solution with 70% ethanol, wipe off 70% ethanol on the surface with paper towels, and bring them into the tissue culture hood.
3. Take the 6-well plates out from the incubator. Carefully pipette the conditioned m....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 mL serological pipettes	Olympus plastics	12-104
15 mL sterile centrifuge tubes	Olympus plastics	28-103
1 mL pipette tip	ART molecular bioproducts	2779-RI
2 mL aspirating pipette	Falcon	357558
24-well plate	Millipore	ECM507	Part of ECM507, or can be purchased separately
4x lysis buffer	Millipore	ECM507	Part of ECM507, or can be purchased separately
5 μm Transwell insert	Millipore	ECM507	Part of ECM507, or can be purchased separately
Accutase	Innovative cell technologies	AT-104
B27	Gibco	12587-010
CyQuant Dye	Millipore	ECM507	Part of ECM507, or can be purchased separately
DMEM	Gibco	11965-092
DMEM:F12	Gibco	10565-018
EGF	Peprotech	AF-100-15
FBS	Gibco	26140
FGF	Peprotech	100-18B
IMDM	Gibco	12440-053
PBS	Gibco	14190-144
Pen Strep	Gibco	15140-122
Trypan blue	Biorad	1450021