Neutrophilic granulocytes are able to release their own DNA as neutrophil extracellular traps (NETs) to capture and eliminate pathogens. DNA expulsion (NETosis) has also been documented for other cells and organisms, thus highlighting the evolutionary conservation of this process. Moreover, dysregulated NETosis has been implicated in many diseases, including cancer and inflammatory disorders. During NETosis, neutrophils undergo dynamic and dramatic alterations of their cellular as well as sub-cellular morphology whose biophysical basis is poorly understood. Here we investigate NETosis in real-time on the single-cell level using fluorescence and atomic force microscopy. Our results show that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status. Entropic chromatin swelling is the major physical driving force that causes cell morphology changes and the rupture of both nuclear envelope and plasma membrane. Through its material properties, chromatin thus directly orchestrates this complex biological process.

Serotonin is an important neurotransmitter involved in various functions of the nervous, blood, and immune system. In general, detection of small biomolecules such as serotonin in real time with high spatial and temporal resolution remains challenging with conventional sensors and methods. In this work, we designed a near-infrared (nIR) fluorescent nanosensor (NIRSer) based on fluorescent singlewalled carbon nanotubes (SWCNTs) to image the release of serotonin from human blood platelets in real time. The nanosensor consists of a nonbleaching SWCNT backbone, which is fluorescent in the beneficial nIR tissue transparency window (800−1700 nm) and a serotonin binding DNA aptamer. The fluorescence of the NIRSer sensor (995 nm emission wavelength for (6,5)-SWCNTs) increases in response to serotonin by a factor up to 1.8. It detects serotonin reversibly with a dissociation constant of 301 nM ± 138 nM and a dynamic linear range in the physiologically relevant region from 100 nM to 1 μM. As a proof of principle, we detected serotonin release patterns from activated platelets on the single-cell level. Imaging of the nanosensors around and under the platelets enabled us to locate hot spots of serotonin release and quantify the time delay (≈ 21−30 s) between stimulation and release in a population of platelets, highlighting the spatiotemporal resolution of this nanosensor approach. In summary, we report a nIR fluorescent nanosensor for the neurotransmitter serotonin and show its potential for imaging of chemical communication between cells.

The formation of neutrophil extracellular traps (NETs) is an immune defense mechanism of neutrophilic granulocytes. Moreover, it is also involved in the pathogenesis of autoimmune, inflammatory, and neoplastic diseases. For that reason, the process of NET formation (NETosis) is subject of intense ongoing research. In vitro approaches to quantify NET formation are commonly used and involve neutrophil stimulation with various activators such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), or calcium ionophores (CaI). However, the experimental conditions of these experiments, particularly the media and media supplements employed by different research groups, vary considerably, rendering comparisons of results difficult. Here, we present the first standardized investigation of the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. In contrast, in murine neutrophils, NETosis was inhibited by FCS and BSA, regardless of the inducer employed. This shows that mouse and human neutrophils have different susceptibilities toward the inhibition of NETosis by albumin or serum components. Furthermore, we provide experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. We also put our results into the context of media supplements most commonly used in NET research. In experiments with human neutrophils, either FCS (0.5–10%), heat-inactivated (hiFCS, 0.1–10%) or human serum albumin (HSA, 0.05–2%) was commonly added to the medium. For murine neutrophils, serum-free medium was used in most cases for stimulation with LPS and CaI, reflecting the different sensitivities of human and murine neutrophils to media supplements. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research.

Integrins are transmembrane receptors that mediate cell-adhesion, signaling cascades and platelet-mediated blood clotting. Most integrins bind to the common short peptide Arg-Gly-Asp (RGD). The conformational freedom of the RGD motif determines how strong and to which integrins it binds. Here, we present a novel approach to tune binding constants by confining RGD peptide motifs via noncovalent adsorption of single-stranded DNA (ssDNA) anchors onto single-walled carbon nanotubes (SWCNTs). Semiconducting SWCNTs display fluorescence in the near-infrared (nIR) region and are versatile fluorescent building blocks for imaging and biosensing. The basic idea of this approach is that the DNA adsorbed on the SWCNT surface determines the conformational freedom of the RGD motif and affects binding affinities. The RGD motif was conjugated to different ssDNA sequences in both linear ssDNA-RGD and bridged ssDNA-RGD-ssDNA geometries. Molecular dynamics (MD) simulations show that the RGD motif in all the synthesized systems is mostly exposed to solvent and thus available for binding, but its flexibility depends on the exact geometry. The affinity for the human platelet integrin αβ could be modulated up to 15-fold by changing the ssDNA sequence. IC values varied from 309 nM for (C)-RGD/SWCNT hybrids to 29 nM for (GT)-RGD/SWCNT hybrids. When immobilized onto surface adhesion of epithelial cells increased 6-fold for (GT)-RGD/SWCNTs. (GT)-RGD/SWCNTs also increased the number of adhering human platelets by a factor of 4.8. Additionally, αβ integrins on human platelets were labeled in the nIR by incubating them with these ssDNA-peptide/SWCNT hybrids. In summary, we show that ssDNA-peptide hybrid structures noncovalently adsorb onto SWCNTs and serve as recognition units for cell surface receptors such as integrins. The DNA sequence affects the overall RGD affinity, which is a versatile and straightforward approach to tune binding affinities. In combination with the nIR fluorescence properties of SWCNTs, these new hybrid materials promise many applications in integrin targeting and bioimaging.