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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (MΦ) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

Abstract

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (MΦ) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study MΦ phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance MΦ phagocytosis of non-opsonized zymosan of both naive and IFN-γ treated MΦ. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Introduction

Mesenchymal stem cells (MSC) are progenitor cells that give rise to connective tissue cells. MSC are present in adult mammalian tissues and can be isolated from the bone marrow1. Due to their immunomodulatory properties, these cells are widely studied2. Early studies focused on MSC regulation of T-cells3,4,5,6 but more recently, their regulation of macrophage cells (MΦ), a major cellular component of innate immunity, has received increased attention7,

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

  1. Preparation of growth medium
    1. For MSC and LADMAC, add 50 mL of FBS and 5 mL of 100x antibiotic/antimycotic mix to 500 mL of high glucose DMEM.
    2. F.......

Representative Results

After calculating mean ± SEM for each group at all time points, the data is presented in line graph format with the Y-axis as the Relative Fluorescent Intensity and the X-axis as Time. Supplementary File 1 provides an example of raw data from a kinetic read of the 96-well plate in a spreadsheet format.

In this study, the optimal results presented in Figure 3A, and Table 3 demonstrate that 1) co-culture with MSC enhances .......

Discussion

Analysis of phagocytosis using bioparticles conjugated to a pH-sensitive dye is a relatively new tool that has proven advantageous over traditional fluorescently labeled particles12,19,20. With traditional fluorescent-labeled particles, only end-point analysis is feasible. Detection is carried out with fluorescent microscopy and/or spectrofluorometry after washing or quenching particles that have not been taken up by the phagocy.......

Acknowledgements

This work was supported by the NSF Major Research Instrument mechanism under grant numbers 1626093 and 1919583.

....

Materials

NameCompanyCatalog NumberComments
96 Well Black Polystyrene MicroplateMilliporeSigmaCLS3603-48EA
0.4% trypan blue solutionMilliporeSigmaT8154-20ML
15 mL and 50 mL Conical Sterile Polypropylene Centrifuge TubesThermoFisher339653
4-well Chambered Coverglass w/ non-removable wellsThermoFisher155382PK
Antibiotic-Antimycotic (100X) GibcoThermoFisher15240096
Axiobserver 7 Imaging SystemZeiss
Bovine Serum Albumin (BSA)MilliporeSigmaA8806-1G
Cell lifterMilliporeSigmaCLS3008-100EA
Culture flasks, tissue culture treated, surface area 75 cm2, canted neck, with cap, filteredMilliporeSigmaC7231-120EA
D1 ORL UVA [D1]ATCCCRL-12424Mouse MSC Cell Line
DMEM, High GlucoseThermoFisher11965092
Fetal Bovine Serum, qualified, heat inactivatedThermoFisher16140071
HemocytometerFisherScientific02-671-51B
I-11.15ATCCCRL-2470Mouse MΦ Cell Line
LADMAC Cell LineATCCCRL-2420LADMAC cells secrete the growth factor colony stimulating factor 1 (CSF-1).
Live-Cell Imaging solutionThermoFisherA14291DJ
PBS, pH 7.4ThermoFisher10010031
pHrodo Green Zymosan Bioparticles ConjugateThermoFisherP35365
Recombinant Murine IFN-γPreprotech315-05
Spectramax i3XMolecular Devices
Sterile Single Use Vacuum Filter Units, 250 mL, 0.2 µmThermoFisher568-0020
Sterile syringe filters, 0.2 micrometerThermoFisher723-2520
Tissue-culture treated culture dishes, 100 mm x 20 mmMilliporeSigmaCLS430167-100EA
Trypsin-EDTA (0.05%), phenol redThermoFisher25300054

References

  1. Phinney, D. G., Prockop, D. J. Concise review: Mesenchymal stem/multipotent stromal cells: The state of transdifferentiation and modes of tissue repair--current views. Stem Cells. 25 (11), 2896-2902 (2007).
  2. Bernardo, M. E., Fibbe, W. E.

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