✍🏼 Justin Langerman, Sevana Baghdasarian, Rene Yu-Hong Cheng, Richard G. James, Kathrin Plath & Dino Di Carlo

🏠 Department of Biological Chemistry, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, CA, USA

📑 Nature Protocols (2025)

Abstract
Cells secrete numerous proteins and other biomolecules into their surroundings to achieve critical functions—from communicating with other cells to blocking the activity of pathogens. Secretion of cytokines, growth factors, extracellular vesicles and even recombinant biologic drugs defines the therapeutic potency of many cell therapies. However, gene expression states that drive specific secretory phenotypes are largely unknown. We provide a protocol that enables the secretion amount of a target protein encoded (SEC) by oligonucleotide barcodes to be linked with transcriptional sequencing (seq) for thousands of single cells. SEC-seq leverages microscale hydrogel particles called Nanovials to isolate cells and capture their secretions in close proximity, oligonucleotide-labeled antibodies to tag secretions on Nanovials and flow cytometry and single-cell RNA-sequencing (scRNA-seq) platforms for readout. Cells on Nanovials can be sorted on the basis of viability, secretion amount or other surface markers without fixation or permeabilization, and cell- and secretion-containing Nanovials are directly introduced into microfluidic droplets-in-oil emulsions for single-cell barcoding of cell transcriptomes and secretions. We have used SEC-seq to link T cell receptor sequences to the relative amount of associated cytokine secretions, surface marker gene expression with a highly secreting and potential regenerative population of mesenchymal stromal cells and the transcriptome with high immunoglobulin secretion from plasma cells. Nanovial modification and cell loading takes <4 h, and once the desired incubation time is over, staining, cell sorting and emulsion generation for scRNA-seq can also be completed in <4 h. Compared to related techniques that link secretions to a cell’s surface, SEC-seq provides a general solution across any secretion target because of the ease with which biotinylated Nanovials can be modified. By linking gene expression and secretory strength, SEC-seq can expand our understanding of cell secretion, how it is regulated and how it can be engineered to make better therapies.

How the WOLF was used in this study
The authors describe a workflow in which flow cytometric sorting of living cells on functionalized Nanovials is a key step in the SEC-seq protocol. In this method, cells are first captured on microscale hydrogel Nanovials that retain both individual cells and their secreted proteins in close proximity. After incubation and staining, cells on Nanovials are sorted based on viability, secretion amount, or specific surface markers using flow cytometry (as described in the protocol), allowing selection of defined functional subpopulations without fixation or permeabilization. These sorted Nanovials—each bearing a single cell and its secretions—are then introduced into a microfluidic droplet platform for barcoding and single-cell RNA sequencing, linking each cell’s transcriptome with its secretion phenotype in downstream analysis. By integrating sorting with scRNA-seq library preparation, the WOLF-style sorting step ensures that only the desired live, functional cells contribute to the linked transcriptomic and secretomic dataset.