Protocol for localized macrophage stimulation with small-molecule TLR agonist via fluidic force microscopy

Elizabeth J Mulder; Brittany Moser; Jennifer Delgado; Rachel Steinhardt; Aaron P Esser-Kahn

doi:10.1016/j.xpro.2024.102873

Protocol for localized macrophage stimulation with small-molecule TLR agonist via fluidic force microscopy

STAR Protoc. 2024 Mar 15;5(1):102873. doi: 10.1016/j.xpro.2024.102873. Epub 2024 Feb 29.

Authors

Elizabeth J Mulder¹, Brittany Moser², Jennifer Delgado², Rachel Steinhardt², Aaron P Esser-Kahn³

Affiliations

¹ Department of Physics, University of Chicago, Chicago, IL 60637, USA. Electronic address: ejmulder@uchicago.edu.
² Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL 60637, USA.
³ Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL 60637, USA. Electronic address: aesserkahn@uchicago.edu.

Abstract

Here, we present a protocol to deliver nanoliter volumes of Toll-like receptor (TLR) agonist onto a culture of nuclear factor κB (NF-κB) reporter macrophages using fluidic force microscopy and a micron-scale probe. We describe steps for quantifying the dose of agonist by modeling their diffusion with experimental inputs. We then detail procedures for quantifying and categorizing macrophage responses to individual and varied doses and combining agonist concentration and macrophage response to analyze the NF-κB response to localized TLR stimulation. For complete details on the use and execution of this protocol, please refer to Mulder et al. (2024).¹.

Keywords: AFM; Atomic Force Microscopy; Biophysics; Cell Biology; Cell culture; Cell-based Assays; Immunology; Microscopy; Single Cell.

MeSH terms

Macrophages
Microscopy, Atomic Force
NF-kappa B* / physiology
Toll-Like Receptor 4
Toll-Like Receptors*

Substances

NF-kappa B
Toll-Like Receptors
Toll-Like Receptor 4