Parallel High-Resolution Imaging of Leukocyte Chemotaxis Under Agarose with Rho-Family GTPase Biosensors

Abstract

Neutrophils are key early responders in the innate immune response that use chemotaxis, the directed migration along chemical gradients, to reach sites of infection or inflammation. This process requires integrating inputs from cell surface receptors with the cell's polarity and motility signaling network, in which highly dynamic and interconnected signaling by Rho-family GTPases plays a central role. To understand this fundamentally important behavior, we describe a high-resolution, under-agarose chemotaxis assay for use with neutrophil-like cell lines (HL-60 or PLB-985) or with primary neutrophils. We also describe how to use optical uncaging of chemoattractants to stimulate cells in this assay. These techniques are compatible with epifluorescence, total internal reflection fluorescence (TIRF), and confocal microscopy. Additionally, we cover how to measure the activities of Rho-family GTPases in this context using Förster resonance energy transfer (FRET)-based biosensors. The specific experimental steps outlined in this chapter include how to (1) set up the under-agarose assay, (2) optically pattern chemoattractant gradients, (3) image cells, and (4) conduct basic image analysis for FRET biosensors.

WASP integrates substrate topology and cell polarity to guide neutrophil migration

“…For all experiments except the STED, collagen, nanopatterns, and EDTA treatment, HL-60s were prepared via a standard under-agarose protocol ( Bell et al, 2018 ). For this work, a solution of 2% low-melt agarose (A-204; Gold Biotechnology) was made in L-15 media (21083-027; Gibco BRL) and microwaved in a loosely capped conical tube placed in a water reservoir.…”

Optogenetic control of receptors reveals distinct roles for actin- and Cdc42-dependent negative signals in chemotactic signal processing

“…Several experiments were conducted using an under-agarose cell preparation on a 96-well plate format (Cellvis Cat#: P96-1.5H-N). A detailed protocol for the under-agarose preparation can be found as described 44 . In brief, differentiated cells (~1,000 -1,500) were plated in the center of a well in a 5 µL drop of 2% FBS + L-15 imaging media.…”

WASP integrates substrate topology and cell polarity to guide neutrophil migration

“…For all experiments excepting the STED, collagen, nanopatterns, and EDTA treatment, HL-60s were confined based on the method described in (Bell et al, 2018). For this work, a solution of 2% low-melt agarose (Gold Biotechnology, A-204) was made in L-15 media (Gibco, 21083-027) and microwaved in a loosely capped conical placed in a water reservoir.…”