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 Preparation of Conditioned Media Warm up the tumor cell maintenance medium at a 37 °C water bath for 20 min. 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 p…

Materials

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 pipet	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