Flow-cytometric (FC) detection of proteins in single cells is a rapid, quantitative method for single-cell protein analysis. Recent advancements in microfluidic technologies have leveraged miniaturization and automation to adapt flow cytometry for analyzing single cell protein profiles both for cell surface and intracellular proteins. Here, we describe the method for microfluidic FC, along with instructions to build a microfluidic platform capable of automated cell culture, cell surface receptor immunostaining, intracellular phosphoprotein and intracellular cytokine immunostaining, and analysis using micro-flow cytometry. As a demonstration of our platform and protocol, we detail the profiling of TLR4 receptor activation, ERK1/2 phosphorylation, and TNFα production in LPS stimulated macrophages using the microfluidic platform.
Keywords: Cell surface receptors; Cytokine; Flow cytometry; Immunostaining; Microfluidic; Phosphoprotein; Single-cell resolution.