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).

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