In vitro Assessment of Efferocytic Capacity of Human Macrophages Using Flow Cytometry

Abstract

Clearance of dying cells, named efferocytosis, is a pivotal function of professional phagocytes that impedes the accumulation of cell debris. Efferocytosis can be experimentally assessed by differentially tagging the target cells and professional phagocytes and analyzing by cell imaging or flow cytometry. Here, we describe an assay to evaluate the uptake of apoptotic cells (ACs) by human macrophages in vitro by labeling the different cells with commercially available dyes and analysis by flow cytometry. We detail the methods to prepare and label human macrophages and apoptotic lymphocytes and the in vitro approach to determine AC uptake. This protocol is based on previously published literature and allows for in vitro modeling of the efficiency of AC engulfment during continual efferocytosis process. Also, it can be modified to evaluate the clearance of different cell types by diverse professional phagocytes.

Keywords: Apoptotic cells; Efferocytosis; Flow cytometry; PBMC-derived macrophages; THP-1-derived macrophage.

Figure 1.. Schematics of the gradient obtained by centrifugation to isolate peripheral blood mononuclear cells (PBMCs)

Figure 2.. Flow cytometric analysis to determine the uptake of apoptotic cells by the macrophages.

THP-1-derived macrophages (CTV-labeled) were incubated with UV-irradiated apoptotic Jurkat cells for 18 h (first round, pHRodo-labeled AC1), and subsequently incubated with a second batch of apoptotic Jurkat cells for 2 h (second round, CTFR-labeled AC2), following the schematics described on the Graphic Abstract. (A) Representative flow plot of gating strategy using FMO controls. Macrophages are initially selected, and debris are excluded based on morphology profile on FSC-A vs. SSC-A plot. Next, doublet exclusion is performed on FSC-A vs. FSC-H plot. Next, dead cells were excluded based on staining for viability dye (Zombie-NIR). Next, labeled macrophages (CFSE⁺) were selected. Gating for labeled macrophages that uptake AC on the single uptake or the first round (AC1) is performed using FMO AC1 sample as reference control. Gating for labeled macrophages that uptake AC on the second round (AC2) is performed using FMO AC2. (B) Percentage of macrophages with internalized AC1 and AC2. Boxes represent the mean of four biological replicates and error bars are ± S.E.M. Each biological replicate is shown as a circle. Significance was calculated by Student’s t-test.