Detecting cytokine release from single T-cells

He Zhu, Gulnaz Stybayeva, Jaime Silangcruz, Jun Yan, Erlan Ramanculov, Satya Dandekar, Michael D. George, Alexander Revzin

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69 Citations (Scopus)

Abstract

The cytokine production by leukocytes correlates with body's ability to mount an immune response and therefore has high diagnostic value. In the present study we employed microfabricated surfaces to capture T-cells from minimally processed human blood, arrange these cells into a single cell array, and then detect interferon (IFN)-γ released from individual cells. The fabrication of cell capture surfaces started with coating a silane-modified glass slide with a uniform layer of poly(ethylene glycol) (PEG) hydrogel. The hydrogel-coated slide was lyophilized and then incubated with a mixture of monoclonal anti-IFN-γ and anti-CD4 antibodies (Abs). To define sites for single cell attachment, PEG hydrogel microwells (20 μm diameter) were photolithographically patterned on top of the Ab-containing hydrogel layer. This micropatterning process resulted in fabrication of PEG hydrogel microwells with Ab-decorated bottom and nonfouling walls. To minimize the blood volume requirement and to precisely define shear stress conditions, the engineered surface was enclosed inside a PDMS-based microfluidic device. Introduction of red blood cell (RBC) depleted whole human blood followed by controlled washing led to the isolation of individual CD4 T-cells within PEG microwells. Mitogenic activation and immunofluorescent staining performed inside the microfluidic chamber revealed IFN-γ cytokine signal colocalized with specific T-cells. The device and process presented here will be expanded in the future to enable multiparametric functional analysis of immune cells organized into high density single cell arrays.

Original languageEnglish
Pages (from-to)8150-8156
Number of pages7
JournalAnalytical Chemistry
Volume81
Issue number19
DOIs
Publication statusPublished - Oct 1 2009
Externally publishedYes

ASJC Scopus subject areas

  • Analytical Chemistry

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  • Cite this

    Zhu, H., Stybayeva, G., Silangcruz, J., Yan, J., Ramanculov, E., Dandekar, S., George, M. D., & Revzin, A. (2009). Detecting cytokine release from single T-cells. Analytical Chemistry, 81(19), 8150-8156. https://doi.org/10.1021/ac901390j