An Assay to Study the Regulation of Macrophage Phagocytosis by Mesenchymal Stem Cells

Abstract

Source: Evans, J. F., et al. Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging. J. Vis. Exp. (2021).

This video demonstrates an assay to study mesenchymal stem cell (MSC) regulation of macrophage phagocytosis in a co-culture. Upon co-culturing MSC with macrophages, non-opsonized zymosan particles conjugated to a pH-sensitive fluorophore are added to be phagocytosed by the macrophages. With the increase in phagocytosis, the fluorescence intensity rises, allowing the determination of MSC-mediated regulation of phagocytic activity.

Protocol

NOTE: All medium preparation and cell culture techniques are carried out under aseptic conditions using a biosafety cabinet with laminar flow. All culture incubation steps described are carried out using an incubator designed to maintain an atmosphere of 37 °C, 5% CO2, and 95% humidity. 1. Cell culture Preparation of growth medium For MSC (mesenchymal stem cells) and LADMAC (mouse bone marrow-derived macrophages; it is d…

Representative Results

Figure 1: Overview of the co-culture and phagocytosis assay workflow. An outline of the workflow for quantitative and qualitative analysis of MΦ phagocytosis activity in co-culture with MSC. Supplementary File 1: A representative spreadsheet file of raw kinetic data from an optimal experiment. <a href="https://www.jove.com/files/ftp_upload/21666/2…

Materials

96-well Black Polystyrene Microplate	MilliporeSigma	CLS3603-48EA
0.4% trypan blue solution	MilliporeSigma	T8154-20ML
15 mL and 50 mL Conical Sterile Polypropylene Centrifuge Tubes	ThermoFisher	339653
Antibiotic-Antimycotic (100X) Gibco	ThermoFisher	15240096
Cell lifter	MilliporeSigma	CLS3008-100EA
Culture flasks, tissue culture treated, surface area 75 cm2, canted neck, with cap, filtered	MilliporeSigma	C7231-120EA
D1 ORL UVA [D1]	ATCC	CRL-12424	Mouse MSC Cell Line
DMEM, High Glucose	ThermoFisher	11965092
Fetal Bovine Serum, qualified, heat-inactivated	ThermoFisher	16140071
Hemocytometer	FisherScientific	02-671-51B
I-11.15	ATCC	CRL-2470	Mouse MΦ Cell Line
LADMAC Cell Line	ATCC	CRL-2420	LADMAC cells secrete the growth factor colony stimulating factor 1 (CSF-1).
Live-Cell Imaging solution	ThermoFisher	A14291DJ
PBS, pH 7.4	ThermoFisher	10010031
pH Rhodo Green Zymosan Bioparticles Conjugate	ThermoFisher	P35365
Spectramax i3X	Molecular Devices
Sterile Single-Use Vacuum Filter Units, 250 mL, 0.2 µm	ThermoFisher	568-0020
Sterile syringe filters, 0.2 micrometer	ThermoFisher	723-2520
Tissue-culture-treated culture dishes, 100 mm x 20 mm	MilliporeSigma	CLS430167-100EA
Trypsin-EDTA (0.05%), phenol red	ThermoFisher	25300054