Single platelet variability governs population sensitivity and initiates intrinsic heterotypic responses

Investigations into the nature of platelet functional variety and consequences for homeostasis require new methods for resolving single platelet phenotypes. Here we combine droplet microfluidics with flow cytometry for high throughput single platelet function analysis. A large-scale sensitivity continuum was shown to be a general feature of human platelets from individual donors, with hypersensitive platelets coordinating significant sensitivity gains in bulk platelet populations and shown to direct aggregation in droplet-confined minimal platelet systems. Sensitivity gains scaled with agonist potency (convulxin &gt; TRAP-14&gt;ADP) and reduced the collagen and thrombin activation threshold required for platelet population polarization into pro-aggregatory and pro-coagulant states. The heterotypic platelet response results from an intrinsic behavioural program. The method and findings invite future discoveries into the nature of hypersensitive platelets and how community effects produce population level responses in health and disease.</p

Rapid microfluidic isolation of virally infected primary bronchial epithelial cells for single cell RNA-seq

Single cell RNA sequencing (scRNA-seq) of the bronchial epithelium enables examination of cellular subtypes and their responses to viral infections. Here we present an optimised method for the isolation of virally infected primary bronchial epithelial cells using a commercially available microfluidic device. Using this method single cells can be rapidly isolated with minimal equipment available in most laboratories. Isolation can be carried out inside biological safety cabinets, permitting the use of virally infected cells. Both cell line and primary cells isolated from the device retained sufficient RNA integrity for the generation of short read sequencing compatible cDNA libraries to facilitate single cell RNA-seq

Dual Dean entrainment with volume ratio modulation for efficient droplet co-encapsulation: extreme single-cell indexing

The future of single cell diversity screens involves ever-larger sample sizes, dictating the need for higher throughput methods with low analytical noise to accurately describe the nature of the cellular system. Current approaches are limited by the Poisson statistic, requiring dilute cell suspensions and associated losses in throughput. In this contribution, we apply Dean entrainment to both cell and bead inputs, defining different volume packets to effect efficient co-encapsulation. Volume ratio scaling was explored to identify optimal conditions. This enabled the co-encapsulation of single cells with reporter beads at rates of ∼1 million cells per hour, while increasing assay signal-to-noise with cell multiplet rates of ∼2.5% and capturing ∼70% of cells. The method, called Pirouette coupling, extends our capacity to investigate biological systems