A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions

• Published in: PloS one Vol. 17; no. 10; p. e0274601
• Main Authors: Dao, Long, Zhao, Qingnan, Hu, Jiemiao, Xia, Xueqing, Yang, Qing, Li, Shulin
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 06.10.2022; Public Library of Science (PLoS)
• Subjects: Analysis; Antibodies; Antigens; Automation; Avidin; Binding; Biology and Life Sciences; Biotin; Biotin - metabolism; Calorimetry; Engineering and Technology; Flow rates; Gene expression; Interleukin 12; Lab Protocol; Ligands; Low flow; Lymphocytes; Lymphocytes T; Medicine and Health Sciences; Methods; Microfluidics; Protein expression; Protein-protein interactions; Proteins; Receptors; Research and Analysis Methods; Surface plasmon resonance; Titration; Titration calorimetry; Vimentin; Visualization; United States
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0274601

Receptor-ligand binding has been analyzed at the protein level using isothermal titration calorimetry and surface plasmon resonance and at the cellular level using interaction-associated downstream gene induction/suppression. However, no currently available technique can characterize this interaction directly through visualization. In addition, all available assays require a large pool of cells; no assay capable of analyzing receptor-ligand interactions at the single-cell level is publicly available. Here, we describe a new microfluidic chip–based technique for analyzing and visualizing these interactions at the single-cell level. First, a protein is immobilized on a glass slide and a low-flow-rate pump is used to isolate cells that express receptors that bind to the immobilized ligand. Specifically, we demonstrate the efficacy of this technique by immobilizing biotin-conjugated FGL2 on an avidin-coated slide chip and passing a mixture of GFP-labeled wild-type T cells and RFP-labeled FcγRIIB-knockout T cells through the chip. Using automated scanning and counting, we found a large number of GFP+ T cells with binding activity but significantly fewer RFP+ FcγRIIB-knockout T cells. We further isolated T cells expressing a membrane-anchored, tumor-targeted IL-12 based on the receptor’s affinity to vimentin to confirm the versatility of our technique. This protocol allows researchers to isolate receptor-expressing cells in about 4 hours for further downstream processing.