Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells
Published: November 30, 2023
Abstract
Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.
Protocol
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Seeding of Peritoneal Cells in Channel Slides After pipetting the peritoneal mouse macrophage cell suspension up and down to reduce clumping, pipette 100 µL suspension into the prefilled channel (volume, 100 µL) of a fibronectin-coated channel slide (a 100 µL channel has the dimensions (length x width x height): 50…
Representative Results
Figure 1: Handling of fibronectin-coated channel slides. (A) A channel slide consists of two reservoirs connected by a channel with the dimensions 50 mm x 5 mm 0.4 mm. Channel slides are initially prefilled by applying 1-2 mL medium to one of the two reservoirs and tilting the slide. (B) Caps can be placed onto the reservoirs prior to incubation. The caps can be conveniently used to pump out unwa…
Disclosures
The authors have nothing to disclose.
Materials
| 24 G plastic catheter | B Braun Mesungen AG, Germany | 4254503-01 | Used for peritoneal lavage |
| Hank's buffered salt solution without Ca2+ and Mg2+ | Thermo Fisher Scientific | 14170120 | Used for peritoneal lavage |
| 14 mL polypropylene round bottom tubes | BD Falcon | 352059 | Used to collect peritoneal cells |
| RPMI 1640 medium containing 20 mM Hepes | Sigma-Aldrich | R7388 | Basis medium for assays |
| Heat-inactivated fetal bovine serum | Thermo Fisher Scientific | 10082139 | Used as supplement for RPMI 1640 media |
| 100x penicillin/streptomycin | Thermo Fisher Scientific | 15140122 | Used as supplement for RPMI 1640 media |
| Fibronectin-coated µ-Slide I chambers | Ibidi, Martinsried, Germany | 80102 | Channel slides used for assays |
| µ-Slide (anodized aluminum) rack | Ibidi, Martinsried, Germany | 80003 | Autoclavable stackable rack for channel slides |
| RPMI 1640 medium containing bicarbonate | Sigma-Aldrich | R8758 | Medium for overnight culture |
| N-(2-mercaptopropionyl)glycine | Sigma-Aldrich | M6635 | Scavenger of reactive oxygen species |
| Alexa Fluor 488-conjugated rat (IgG2a) monoclonal (clone BM8) anti-mouse F4/80 antibody | Thermo Fisher Scientific | MF48020 | Mouse macrophage marker and plasma membrane label |
| CellMask Orange | Thermo Fisher Scientific | C10045 | Red fluorescent plasma membrane stain |
| Succinimidyl ester of pHrodo | Thermo Fisher Scientific | P36600 | Amine-reactive succinimidyl ester of pHrodo |
| Mouse (IgG2b) monoclonal (clone HIR2) anti-human CD235a | Thermo Fisher Scientific | MA1-20893 | Used to opsonize human red blood cells with IgG |
| Alexa Fluor 594-conjugated goat anti-mouse (secondary) IgG antibody | Abcam | Ab150116 | Used to confirm opsonization of human red blood cells with mouse IgG |
| Rat anti-mouse C3b/iC3b/C3c antibody | Hycult Biotech | HM1065 | Used to confirm C3b/iC3b opsonization of human red blood cells |
| Alexa Fluor 488-conjugated goat anti-rat IgG antibody | Thermo Fisher Scientific | A-11006 | Used as secondary antibody to confirm C3b/iC3b opsonization |
| UltraVIEW Vox 3D live cell imaging system | Perkin Elmer, Rodgau, Germany | Spinning disk confocal microscope system | |
| Nikon Eclipse Ti inverse microscope | Nikon, Japan | Inverted microscope | |
| CSU-X1 spinning disk scanner | Yokogawa Electric Corporation, Japan | Nipkow spinning disk unit | |
| 14-bit Hamamatsu C9100-50 Electron Multiplying-Charged Couple Device (EM-CCD) peltier-cooled camera | Hamamatsu Photonics Inc., Japan | EM-CCD camera of the spinning disk confocal microscope system | |
| 488 nm solid state laser, 50 mW | Perkin Elmer, Rodgau, Germany | Laser (488 nm) source of spinning disk confocal microscope system | |
| 561 nm solid state laser, 50 mW | Perkin Elmer, Rodgau, Germany | Laser (561 nm) source of spinning disk confocal microscope system |
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Cite This Article
Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells. J. Vis. Exp. (Pending Publication), e21782, doi: (2023).