Cell Biology

Extracellular vesicles (EVs) are critical mediators of cell–cell communication and play a key role in male reproductive biology by modulating sperm function. This protocol describes a robust and reproducible workflow for isolating EVs from ram seminal plasma using size-exclusion chromatography (SEC) and assessing their uptake by ram spermatozoa. In contrast to ultracentrifugation-based methods, SEC provides a gentle and more efficient isolation approach that preserves EV integrity and functionality. A central innovation of this protocol is the use of carboxyfluorescein succinimidyl ester (CFSE)-labeled seminal plasma EVs (SP-EVs) to evaluate their incorporation into sperm cells through two complementary detection platforms: (i) flow cytometry with standard resolution and (ii) confocal microscopy, for spatial confirmation of EV–sperm interactions. By bridging the gap between EV isolation and functional analysis, this protocol provides a valuable tool for investigating the role of EV–cell interactions. Specifically, it offers potential applications in male fertility preservation, biomarker discovery, and the development of EV-based therapeutic strategies in reproductive medicine.

CD8⁺CD28⁻ T suppressor cells (Ts) have been documented to promote immune tolerance by suppressing effector T cell responses to alloantigens following transplantation. The suppressive function of T cells has been defined as the inhibitory effect of Ts on the proliferation rate of effector T cells. ³H-thymidine is a classical immunological technique for assaying T cell proliferation but this approach has drawbacks such as the inconvenience of working with radioactive materials. Labeling T cells with CFSE allows relatively easy tracking of generations of proliferated cells. In this report, we utilized antigen presenting cells (APCs) and T cells matched for human leukocyte antigen (HLA) class I or class II to study CD8⁺CD28^- T cell suppression generated in vitro by this novel approach of combining allogeneic APCs and γc cytokines. The expanded CD8⁺CD28^- T cells were isolated (purity 95%) and evaluated for their suppressive capacity in mixed lymphocyte reactions using CD4⁺ T cells as responders. Here, we present our adapted protocol for assaying the Ts allospecific suppression of CFSE-labeled responder T cells.

Connexins are membrane bound proteins that facilitate direct and local paracrine mediated cell-to-cell communication through their ability to oligomerise into hexameric hemichannels. When neighbouring channels align, they form gap-junctions that provide a direct route for information transfer between cells. In contrast to intact gap junctions, which typically open under physiological conditions, undocked hemichannels have a low open probability and mainly open in response to injury. Hemichannels permit the release of small molecules and ions (approximately 1kDa) into the local intercellular environment, and excessive expression/activity has been linked to a number of disease conditions. Carboxyfluorescein dye uptake measures functional expression of hemichannels, where increased hemichannel activity/function reflects increased loading. The technique relies on the uptake of a membrane-impermeable fluorescent tracer through open hemichannels, and can be used to compare channel activity between cell monolayers cultured under different conditions, e.g. control versus disease. Other techniques, such as biotinylation and electrophysiology can measure cell surface expression and hemichannel open probability respectively, however, carboxyfluorescein uptake provides a simple, rapid and cost-effective method to determine hemichannel activity in vitro in multiple cell types.

Graphic abstract


Using dye uptake to measure hemichannel activity