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.
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
2. Seed MSC in experimental dishes, Day 1
NOTE: Prior to seeding the MSC, design the experimental 96-well plate layout for fluorescent spectrophotometry and the chamber slide for dynamic imaging. For the 96-well plate, flank experimental wells with wells containing cells but do not use them in the assay. Mark at least four wells to be used for the reagent blank (RBL). See Table 1 for an example 96-well template and Table 2 for an example of a 4-well chamber slide template. Black or white 96-well plates with clear bottoms are recommended. A 1.5 mm borosilicate glass chamber slide is optimal, but the number of chambers can be adjusted depending on the study design. A summary flow chart of the methods that follow is included in Figure 1.
3. Isolate the MΦ and prepare the co-cultures, Day 2
4. Phagocytosis assay, kinetic fluorescent 96-well plate read, Day 3
Table 1: Example of a 96-well plate design. CBL – Cell blank; MSC – seeded day 1; MΦ+ treated and MΦ untreated seeded day 2; RBL reagent blank added on assay day 3.
1 | 2 | 3 | 4 | 5 | 6 | 7 | |
A | CBL | MSC/MΦ | MSC/MΦ+ | MΦ | MΦ+ | CBL | RBL |
B | CBL | MSC/MΦ | MSC/MΦ+ | MΦ | MΦ+ | CBL | RBL |
C | CBL | MSC/MΦ | MSC/MΦ+ | MΦ | MΦ+ | CBL | RBL |
D | CBL | MSC/MΦ | MSC/MΦ+ | MΦ | MΦ+ | CBL | RBL |
E | CBL | MSC/MΦ | MSC/MΦ+ | MΦ | MΦ+ | CBL | RBL |
F | CBL | MSC/MΦ | MSC/MΦ+ | MΦ | MΦ+ | CBL | RBL |
G | CBL | MSC/MΦ | MSC/MΦ+ | MΦ | MΦ+ | CBL | RBL |
H | CBL | MSC/MΦ | MSC/MΦ+ | MΦ | MΦ+ | CBL | RBL |
Table 2: Example of a 4-well chamber slide design. MSC – plated day 1; MΦ+ treated and MΦ untreated plated day 2.
1 | 2 | 3 | 4 |
MSC/MΦ | MSC/MΦ+ | MΦ | MΦ+ |
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. Please click here to download this File.
The authors have nothing to disclose.
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 |