Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Summary

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Abstract

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Introduction

Our body generates 1-10 billion apoptotic cells on a daily basis. Such a large number of apoptotic cells must be cleared in a way that the immune responses remain quiescent. To ensure the clearance of apoptotic cells in a timely manner, numerous types of tissue resident cells and circulating cells develop mechanisms to engulf apoptotic cells¹. Dysfunctional regulation of apoptosis has been implicated in the onset and progression of various inflammatory disease and autoimmunity². Apoptosis also plays a critical role in the pathogenesis of cancer development and its subsequent resistance to conventional treatments

Protocol

Experimental mice were bred and maintained in our mice colony. All animal work was conducted according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati.

1. Preparation of CFSE labeled apoptotic thymocytes

1. Euthanize two naïve C57/B6 mice by CO₂ inhalation for 10 min and dissect to open the chest cavity, remove (pull out) the thymus with curved fine-tip forceps into tissue culture petri dish containing 10 mL of RPMI1640 medium.
2. Obtain single cell suspension by grinding the whole thymus against two frosted ends of the microscope slides and then fi....

Results

Analysis of peritoneal macrophage-mediated engulfment of apoptotic thymocytes. Peritoneal macrophages and apoptotic cells were prepared and co-cultured as described in the protocol. Macrophages were detached and stained with PE conjugated anti-CD11b antibody for 20 min on ice. Macrophages were then washed and processed in a flow cytometer. As seen, there is no CFSE positive macrophage in the bottom right quadrant when no apoptotic cells were added into the culture (Figure 1<.......

Discussion

Apoptosis is a highly conserved cell death process that involves many signal cascades and induces protein expression, secretion, and transportation. Apoptosis is often associated with cellular morphology changes¹⁷. Apoptotic cells actively release cytokines and chemokines that attract phagocytes to migrate to the site and initiate the process of engulfment, an extremely complex pathway under tight control¹⁸. On the other hand, necrotic cell death releases danger signals tha.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ack lysing buffer	GIBCO	A10492
Annexin V/7-AAD	BD Pharmingen	559763
Anti-Mer antibody	R&D Systems	BAF591
CD11b-PE (clone M1/70)	BD Pharmingen	553311
CFSE	Invitrogen	C1157
DMSO	Sigma-Aldrich	D-2650
EDTA (0.5 mM)	GIBCO	15575-020
FACS tubes	BD Biosciences	352017
Frosted slides	Fisher Scientific	12-552-343
Horse Serum (Heat-inactivated)	Invitrogen	26050088
Lidocaine	Sigma-Aldrich	L-5647	Prepare 1% buffer in 1x PBS
PBS, 1x	Corning	21040CV
RPMI-1640	Corning	10040CV
RXDX-106	Selleck Chemicals	CEP-40783
Staurosprine (100mg)	Fisher Scientific	BP2541-100	Add 214.3 ml of DMSO into 100mg to make 1mM stocking solution
Thioglycolate Medium Brewer Modified	BD Biosciences	243010	Prepare 3% thioglycolate buffer in 1´PBS, autoclaved, and store in the dark for 3 months.