Study of Phagolysosome Biogenesis in Live Macrophages

Abstract

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Introduction

Professional phagocytes, including macrophages, play a critical in the immune system. In addition to being the first line of defense in the innate immune system, they also play a critical role in activation of the adaptive immunity through their signaling and antigen presenting role^1-3. While the function of professional phagocytes is multifaceted, phagosome maturation is the critical backbone of the bactericidal and antigen processing function of the professional phagocytes^4,5. Upon the receptor mediated engulfment and uptake of a phagocytic target, such as bacteria, the nascent phagosome goes through a complex and well-orchestrated sequence of ....

Protocol

Animal care and all procedures in this protocol follow the institutional and national guidelines.

Prepare the preparations that are indicated by "Π-" beforehand.

1. Preparation of BMMs

1. Perform the culling of the mice by cervical dislocation.
2. Remove femur and tibia bones, free bones from tissue and trim both ends for the accessibility of the bone marrow.
3. Keep bones in ice-cold PBS or ice-cold DMEM medium (supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, on ice until the next step.
   Note: Conduct the following steps under a cl....

Results

The correct preparation of the cells and the beads for imaging is critical. Figure 1 shows the outline of the seeding, probe-loading and initial imaging steps in this method, based on our optimized protocol for BMMs. It is essential therefore that the protocol parameters be tested and optimized depending on the type of cells and probes that are to be used.

After addition of the IgG-coated beads to the preloaded cells, it is important that the field of view is chosen in a manne.......

Discussion

In the following section we will discuss critical steps of the presented method and its limitations. Further, we will connect some of the common problems with their solutions, introduce possible modifications and consider advantages of our method as well as suitable complementary methods for this approach.

The preparation of the beads, including the coupling of IgG to the bead surface, is crucial and the use of unfresh preparations should be avoided. In additions to that, it is also critical t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagles Medium (D-MEM)	PAA, Austria	E15-009
Dulbecco's Modified Eagles Medium without phenol red (D-MEM)	PAA, Austria	E15-047
Fetal Bovine Serum (FBS)	PAA, Austria	A15-151
L-Glutamine	PAA, Austria	M11-004
PBS	PAA, Austria	H15-002
Penicillin/Streptomycin	PAA, Austria	P11-010
WillCo-dish® Glass bottom dish (35 mm ∅, #1.5)	WillCo Wells, Netherlands	GWSt-3522
CELLviewTM glass bottom dish, 35 mm	Greiner Bio one, Germany	627871	Alternative: Available as segmented
Carboxylated latex microspheres	Polysciences, USA	#09850	Available in different diameters, we used 3 μm
Polystyrene microparticles	Kisker Biotech, Germany	PPs-3.0COOH
Triton-X 100	ROTH, Germany	305.1.3
mouse whole IgG	Rockland, USA	010-0102
EDAC crosslinker	Sigma-Aldrich, USA	E6383-1g
10 mM Tris	Sigma-Aldrich,USA	T1503
1-ml syringe Omnifix -F	B.Braun, Germany	9161406V
26 G needle (0.45*25 mm)	B.Baun, Germany	4657683
RPMI 1640	PAA, Austria	E15-039
Horse Serum	Gibco, USA	16050-122
MES hydrate	Sigma-Aldrich, USA	M2933
0.2 μM syringe mounted filter	Sartorius, Germany	83.1826.001