Calcium Imaging of Non-adherent Cells

Live-cell imaging can reveal dynamic and multimodal cell signaling by monitoring calcium flux. Spatiotemporal changes in Ca2+ concentrations instigate specific downstream processes and by categorizing these events, we can examine the language cells use to communicate both to themselves and with each other. Thus, calcium imaging is an understandably popular and versatile technique that relies on high-resolution optical data as measured by fluorescence intensity. This is executed with relative ease on adherent cells, as changes in fluorescence intensity can be monitored over time in fixed regions of interest. However, perfusion of non-adherent or mildly adherent cells leads to their mechanical displacement thereby hindering the spatial resolution of fluorescence intensity changes through time. Here we provide details of a simple and cost-effective protocol using gelatin to prevent cell dislodgement during the solution exchanges that occur during recording

Nicotinic acetylcholine receptors: Key targets for attenuating neurodegenerative diseases

Nicotinic acetylcholine receptors (nAChRs) are master regulators of immune functions via the cholinergic anti-inflammatory pathway and are expressed in microglia, the brain\u27s resident immune cells. There is an extensive dialogue between the neurons and the glial cells around them from which microglia are tasked with monitoring, nurturing, and defending their microenvironment. Dysregulation of any of these processes can have devastating and long-lasting consequences involving microglia-mediated neuroinflammation associated with neurodegenerative diseases such as Alzheimer\u27s disease, Parkinson\u27s disease, and Huntington\u27s disease, amongst others. Disease-associated microglia acquire a distinguishing phenotype that emphasizes scavenging and defence functions while nurturing and repairing functions become muted. Attempts to resolve this critical imbalance remain a key focus of research. Furthermore, cholinergic modulation of neuroinflammation represents a promising avenue for treatment

Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy