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A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions
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 interactio...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9536614/ https://www.ncbi.nlm.nih.gov/pubmed/36201506 http://dx.doi.org/10.1371/journal.pone.0274601 |
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author | Dao, Long Zhao, Qingnan Hu, Jiemiao Xia, Xueqing Yang, Qing Li, Shulin |
author_facet | Dao, Long Zhao, Qingnan Hu, Jiemiao Xia, Xueqing Yang, Qing Li, Shulin |
author_sort | Dao, Long |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-9536614 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-95366142022-10-07 A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions Dao, Long Zhao, Qingnan Hu, Jiemiao Xia, Xueqing Yang, Qing Li, Shulin PLoS One Lab Protocol 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. Public Library of Science 2022-10-06 /pmc/articles/PMC9536614/ /pubmed/36201506 http://dx.doi.org/10.1371/journal.pone.0274601 Text en https://creativecommons.org/publicdomain/zero/1.0/This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 (https://creativecommons.org/publicdomain/zero/1.0/) public domain dedication. |
spellingShingle | Lab Protocol Dao, Long Zhao, Qingnan Hu, Jiemiao Xia, Xueqing Yang, Qing Li, Shulin A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions |
title | A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions |
title_full | A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions |
title_fullStr | A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions |
title_full_unstemmed | A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions |
title_short | A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions |
title_sort | microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions |
topic | Lab Protocol |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9536614/ https://www.ncbi.nlm.nih.gov/pubmed/36201506 http://dx.doi.org/10.1371/journal.pone.0274601 |
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