Relative Quantitation of Polymerized Actin in Suspension Cells by Flow Cytometry

Background

Biologists have long studied actin polymers using biochemical assays and fluorescence microscopy. The latter has been facilitated by the discovery of phalloidin, a small molecule toxin produced by poisonous mushrooms that stabilizes actin structures by specifically binding adjacent monomers within actin polymers (Vandekerckhove et al., 1985). Fluorescent phalloidin conjugates stain complex actin structures and facilitate detailed visualization by fluorescence microscopy. However, quantitating this fluorescence can be painstaking, particularly when tracing population-level changes. In contrast, flow cytometry quantitates the total fluorescence intensity of thousands of cells per minute.

Here we describe how we estimate differences in the amount of actin polymer between cell populations by staining cells with fluorescent phalloidin, measuring the total cell fluorescence of individual cells using flow cytometry, and finally comparing the fluorescence values of the populations. It is important to note that this approach is comparative and cannot be used to measure the absolute concentrations of cellular actin polymer. This approach requires cells to be in suspension; if your cells require adhesion for proper actin polymerization, we suggest using quantitative fluorescence microscopy as described above.

Although rapid and quantitative, flow cytometry provides no information about cell morphology. Therefore, we routinely save an aliquot of stained cells for visualization using fluorescence microscopy. We highly recommend doing this to ensure proper preservation of your structures of interest (Figure 1). Fluorescence microscopy can also reveal variation in experimental samples that are not apparent by flow cytometry.

This protocol is optimized for differentiated HL-60 cells that can be stimulated with fMLP to induce rapid actin polymerization (Figure 1, for additional information about these cells, see Fritz-Laylin et al., 2017), it can be modified for use with a wide variety of cell types by testing a variety of fixation conditions and buffers. For example, we have adapted this protocol for chytrid fungi (Batrachochytrium dendrobatidis), and the amoeba Naegleria gruberi (unpublished). To do this, we chose conditions that: 1) best preserve the overall morphology reasonably similar to live cells as visualized using DIC or phase contrast microscopy; 2) keep the polymerized actin intact through fixation; 3) when imaged by fluorescence microscopy, stain the appropriate structures. For information on buffers and fixatives (see Note 1) commonly used to preserve actin structures, we recommend the methods resource Fluorescence Procedures for the Actin and Tubulin Cytoskeleton in Fixed Cells from the Mitchison lab at Harvard University (L. Cramer and A. Desai).


Figure 1. An example of actin polymerization in response to cell stimulation. Maximum projection of AlexaFluor^® 488 phalloidin stained HL-60 cells before and after treatment with 20 nM fMLP, which induces actin polymerization and results in brighter cells.