Measurement and quantification of macrophage lytic cell death by kinetic microscopy

Macrophages are innate immune cells that are critical in the maintenance of tissue homeostasis and defense against pathogens. Programmed cell death is a critical tool macrophages use to clear pathogens and to alert surrounding cells to damage following induction of cell death. Diverse forms of cell death, including pyroptosis, necroptosis, ferroptosis, and secondary apoptosis following apoptosis, result in loss of plasma membrane integrity. Membrane disruption occurs following the oligomerization of pore-forming proteins into the cell membrane, releasing cytokines and damage associated molecular patterns that trigger the immune response. Thus, quantification of cell membrane permeability is an effective method for assessing cell death in macrophages. Many different factors, including macrophage polarization, stimulation, and inflammatory state, can impact macrophage predisposition to, and rate of, programmed cell death. Thus, it is important to have a method of accurately assessing macrophage cell death kinetically, rather than at a single endpoint. In this protocol, we describe a protocol for assessing cell death in macrophages by quantifying cell membrane permeability using kinetic microscopy. This method overcomes limitations of common single time point metrics for assessing cell death and is adaptable and scalable for use in assessing cell death across different cell types and treatment conditions.