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Cell Biology

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Protocols in Past Issues

A Versatile In Vitro Quantitative Assay for Macrophage Efferocytosis in Diverse Research Applications
XX Xiaoge Xu
TS Tong Su
QS Qinuo Sun
XW Xiaoou Wang
YL Yanjie Liu
HW Huihui Wang
Q&A 0 Q&A
543 Views
May 20, 2026

Macrophage efferocytosis is a previously unrecognized key pathogenic event, engulfing apoptotic targets, preventing inflammation and necrosis, and maintaining immune homeostasis. The phagocytic function can be disrupted by harmful factors and toxic substances. This protocol describes a versatile visualized in vitro method that can be used for the detection of general efferocytosis. This method is applicable to a wide range of research scenarios. As a representative application, it can be used to evaluate macrophage efferocytosis dysfunction in diseases linked to harmful exposures, including atherosclerosis, chronic inflammation, and malignant tumors. Among them, the detection of the effects of oxidized low-density lipoprotein (ox-LDL) and arsenite on macrophage efferocytosis capacity is an exemplary application of this protocol. Primary macrophages collected from mice were labeled with a cell-tracking dye and exposed to ox-LDL or arsenite, then co-cultured with apoptotic thymocytes or hepatocytes (labeled with another cell-tracking dye) for 2 h at a ratio of 5:1. Macrophage efferocytosis was visualized using a laser confocal microscope. The results indicate that arsenite impaired macrophage efferocytosis, leading to insufficient clearance of apoptotic thymocytes or hepatocytes. This method can be extended to subsequent studies, including those involving different types of phagocytes, apoptotic cell models, and research related to exposure to various factors.

Dissecting the Mechanical Control of Mitotic Entry Using a Cell Confinement Setup
MD Margarida Dantas
DV Débora Vareiro
JF Jorge G. Ferreira
Q&A 0 Q&A
2868 Views
Mar 20, 2024

Proliferating cells need to cope with extensive cytoskeletal and nuclear remodeling as they prepare to divide. These events are tightly regulated by the nuclear translocation of the cyclin B1-CDK1 complex, that is partly dependent on nuclear tension. Standard experimental approaches do not allow the manipulation of forces acting on cells in a time-resolved manner. Here, we describe a protocol that enables dynamic mechanical manipulation of single cells with high spatial and temporal resolution and its application in the context of cell division. In addition, we also outline a method for the manipulation of substrate stiffness using polyacrylamide hydrogels. Finally, we describe a static cell confinement setup, which can be used to study the impact of prolonged mechanical stimulation in populations of cells.


Key features

• Protocol for microfabrication of confinement devices.

• Single-cell dynamic confinement coupled with high-resolution microscopy.

• Static cell confinement protocol that can be combined with super-resolution STED microscopy.

• Analysis of the mechanical control of mitotic entry in a time-resolved manner.


Graphical overview


Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry
MS Michael S. Schappe
BD Bimal N. Desai
Q&A 0 Q&A
10680 Views
Jul 20, 2018
After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways–one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe et al., 2018). The assay relies on stimulating the cells with LPS and measuring the cell surface levels of TLR4 (or CD14) at various time points using flow cytometry. Although we detail the method specifically for TLR4 and CD14 from murine bone marrow-derived macrophages, it can be readily adapted to evaluate receptor endocytosis in a variety of other signaling contexts.

DQ-Red BSA Trafficking Assay in Cultured Cells to Assess Cargo Delivery to Lysosomes
Rituraj Marwaha Rituraj Marwaha
Mahak Sharma Mahak Sharma
Q&A 1 Q&A
26782 Views
Oct 5, 2017
Lysosomes are the terminal end of the endocytic pathway having acidic environment required for active hydrolases that degrade the cargo delivered to these compartments. This process of cargo delivery and degradation by endo-lysosomes is a tightly regulated process and important for maintaining cellular homeostasis. Cargos like EGF (Epidermal Growth Factor), Dil-LDL (3,3’-Dioctadecylindocarbocyanine-Low Density Lipoprotein), Dextran, DQ-BSA (Dye Quenched-Bovine Serum Albumin) etc., are routinely used by researchers to analyze the role of various proteins in endocytic pathway. Trafficking of DQ-BSA in cells depleted of or over-expressing the gene of interest is a useful assay for identifying the role of various proteins in endocytic trafficking pathway. The protocol describes the DQ-Red BSA trafficking assay that can be used to study endocytic trafficking in various cell types.

Influenza Virus-cell Fusion Inhibition Assay
Mayo Yasugi Mayo Yasugi
Kazuyoshi Ikuta Kazuyoshi Ikuta
Q&A 1 Q&A
11517 Views
Dec 20, 2013
During viral infection to host cells, several viruses undergo the process of endocytosis and pH-dependent fusion. By fusion of viral membrane with host cellular membrane, the viral core invades to host cytoplasm. A part of monoclonal antibodies against viral membrane protein have potential to inhibit the viral fusion step. Here we describe in vitro influenza virus-cell fusion inhibition assay. The infected cells expressing viral membrane protein, such as hemagglutinin (HA), on cellular surface are incubated with monoclonal antibodies targeting viral membrane protein. Then the cells are incubated under low pH condition. If the antibody does not inhibit the fusion step, we can see multinucleated giant cells.

Assessment of Human Dendritic Cell Antigen Uptake by Flow Cytometry
AL Ana Luque
SC Sonia Cárdenas-Brito
RO Rut Olivar
JA Josep M. Aran
Q&A 0 Q&A
19118 Views
Nov 20, 2013
Antigen uptake by dendritic cells is the first key step towards induction of antigen-specific T-cell responses. This flow cytometry-based protocol describes the analysis of dendritic cell uptake of soluble antigens through two different mechanisms: non-specific macropinocytosis (using Lucifer Yelloy CH), and receptor-mediated endocytosis (using DQTM Ovalbumin). The protocol is generated based on data presented in Olivar et al. (2013).

Epidermal Growth Factor (EGF) Receptor Endocytosis Assay in A549 Cells
SR Sabrina Rizzolio
LT Luca Tamagnone
Q&A 0 Q&A
19455 Views
Aug 5, 2013
The following endocytosis assay has been optimized to assess EGF-stimulated EGFR endocytosis; but could be modified to assess other ligand-stimulated endocytosis of plasma membrane receptors (for which fluorochrome-conjugated ligands are available to track their receptor internalization). In brief, cells are treated with fluorescent EGF at 4 °C to allow binding to the receptor, but not internalization; then, endocytosis is allowed at 37 °C for different timepoints. For the setting up of this protocol we are really indebted to Dr. Letizia Lanzetti (Lanzetti et al., 2000).