Single Cell Analysis of Neutrophils NETs by Microscopic LSPR Imaging System

Abstract

A simple microengraving cell monitoring method for neutrophil extracellular traps (NETs) released from single neutrophils has been realized using a polydimethylsiloxane (PDMS) microwell array (MWA) sheet on a plasmon chip platform. An imbalance between NETs formation and the succeeding degradation (NETosis) are considered associated with autoimmune disease and its pathogenesis. Thus, an alternative platform that can conduct monitoring of this activity on single cell level at minimum cost but with great sensitivity is greatly desired. The developed MWA plasmon chips allow single cell isolation of neutrophils from 150 µL suspension (6.0 × 105 cells/mL) with an efficiency of 36.3%; 105 microwells with single cell condition. To demonstrate the utility of the chip, trapped cells were incubated between 2 to 4 h after introducing with 100 nM phorbol 12-myristate 13-acetate (PMA) before measurement. Under observation using a hyperspectral imaging system that allows high-throughput screening, the neutrophils stimulated by PMA solution show a significant release of fibrils and NETs after 4 h, with observed maximum areas between 314–758 µm2. An average absorption peak wavelength shows a redshift of Δλ = 1.5 nm as neutrophils release NETs.

Neutrophil extracellular traps in adult diseases and neonatal bacterial infectious diseases: A review

“…The earlier processes collectively refer to NETosis, which is called lytic NETs [ 30 ]. NETosis generally takes around 3–4 h to complete [ 31 ]. Additionally, it has been discovered that the rapid formation and release of NETs can occur within 5–60 min without the need for neutrophil death [ 32 ].…”

Targeting Neutrophil Extracellular Traps in Gouty Arthritis: Insights into Pathogenesis and Therapeutic Potential

“…This process only takes 5-60 minutes, which is different from the release of suicidal NOXdependent NETosis or NOX-independent NETosis lasting more than 3 hours. 125 At the end of this process, neutrophils eventually rupture and die. Yipp et al 45,126 proposed that the formation of nuclear DNA in rapid NETosis was dependent on the binding of LPS.…”

Functional blood cell analysis by label-free biosensors and single-cell technologies

“…These methods could prove to be a label-free alternative to current methods applied and developed for NET quantification [172]. In their microwell-based localized surface plasmon resonance based setup Ali et al demonstrated the label-free analysis of single neutrophil granulocytes following PMA treatment [173]. Deep-learning based image analysis based on bright-field microscopic images Hhaung et al were able to classify activated and inactivated neutrophils, with further improvement these efforts could prove to be useful later in image based WBC classification as well [174].…”