Cell Biology

Accurate identification of cell cycle stages is essential for investigating fundamental biological processes such as proliferation, differentiation, and tumorigenesis. While flow cytometry remains a widely used technique for such analyses, it is limited by its lack of single-cell resolution and its requirement for large sample sizes due to its population-based approach. These limitations underscore the need for alternative or complementary methods that offer single-cell precision with compatibility for small-scale applications. We present ImmunoCellCycle-ID, an immunofluorescence-based method that leverages the spatial distribution of endogenous markers, such as DNA, proliferating cell nuclear antigen (PCNA), centromere protein F (CENP-F), and centromere protein C (CENP-C), to reliably distinguish G1, early S, late S, early G2, late G2, and all mitotic sub-stages. This technique does not rely on precise signal quantification and utilizes standard immunofluorescence protocols alongside conventional laboratory microscopes, ensuring broad accessibility. Importantly, ImmunoCellCycle-ID detects endogenous proteins without the need for genetic modification, making it readily applicable to a wide range of human cell lines. Beyond its utility for single-cell resolution, the method can be scaled for population-level analyses, similar to flow cytometry. With its precision, versatility, and ease of implementation, ImmunoCellCycle-ID offers a powerful tool for high-resolution cell cycle profiling across diverse experimental platforms.

Single-cell RNA sequencing has revolutionized molecular cell biology by enabling the identification of unique transcription profiles and cell transcription states within the same tissue. However, tissue dissociation presents a challenge for non-model organisms, as commercial kits are often incompatible, and current protocols rely on tissue enzymatic digestion for extended periods. Tissue digestion can alter cell transcription in response to temperature and the stress caused by enzymatic treatment. Here, we propose a protocol to stabilize RNA using a deep eutectic solvent (Vivophix, Rapid Labs) prior to tissue dissociation, thereby avoiding transcription changes induced by the process and preventing RNase activity during incubation. We validated this methodology for three medically important insect vectors: Anopheles gambiae, Aedes aegypti, and Lutzomyia longipalpis. Single-cell RNA sequencing using our insect midgut dissociation protocol yielded high-quality sequencing results, with a high number of cells recovered, a low percentage of mitochondrial reads, and a low percentage of ambient RNA—two hallmark standards of cell quality.

Formalin-fixed paraffin-embedded (FFPE) samples remain an underutilized resource in single-cell omics due to RNA degradation from formalin fixation. Here, we present snPATHO-seq, a robust and adaptable approach that enables the generation of high-quality single-nucleus (sn) transcriptomic data from FFPE tissues, utilizing advancements in single-cell genomic techniques. The snPATHO-seq workflow integrates optimized nuclei isolation with the 10× Genomics Flex assay, targeting short RNA fragments to mitigate FFPE-related RNA degradation. Benchmarking against standard 10× 3' and Flex assays for fresh/frozen tissues confirmed robust detection of transcriptomic signatures and cell types. snPATHO-seq demonstrated high performance across diverse FFPE samples, including diseased tissues like breast cancer. It seamlessly integrates with FFPE spatial transcriptomics (e.g., FFPE Visium) for multi-modal spatial and single-nucleus profiling. Compared to workflows like 10× Genomics’ snFFPE, snPATHO-seq delivers superior data quality by reducing tissue debris and preserving RNA integrity via nuclei isolation. This cost-effective workflow enables high-resolution transcriptomics of archival FFPE samples, advancing single-cell omics in translational and clinical research.

Bone repair is a complex regenerative process relying on skeletal stem/progenitor cells (SSPCs) recruited predominantly from the periosteum. Activation and differentiation of periosteal SSPCs occur in a heterogeneous environment, raising the need for single cell/nucleus transcriptomics to decipher the response of the periosteum to injury. Enzymatic cell dissociation can induce a stress response affecting the transcriptome and lead to overrepresentation of certain cell types (i.e., immune and endothelial cells) and low coverage of other cell types of interest. To counteract these limitations, we optimized a protocol to isolate nuclei directly from the intact periosteum and from the fracture callus to perform single-nucleus RNA sequencing. This protocol is adapted for fresh murine periosteum, fracture callus, and frozen human periosteum. Nuclei are isolated using mechanical extraction combined with fluorescence-based nuclei sorting to obtain high-quality nucleus suspensions. This protocol allows the capture of the full diversity of cell types in the periosteum and fracture environment to better reflect the in vivo tissue composition.

Single-cell transcriptomic analyses have emerged as very powerful tools to query the gene expression changes at the single-cell level in physiological and pathological conditions. The quality of the analysis is heavily dependent on tissue digestion protocols, with the goal of preserving thousands of single live cells to submit to the subsequent processing steps and analysis. Multiple digestion protocols that use different enzymes to digest the tissues have been described. Harsh digestion can damage certain cell types, but this might be required to digest especially fibrotic tissue as in our experimental condition. In this paper, we summarize a collagenase type I digestion protocol for preparing the single-cell suspension from fibrovascular tissues surgically removed from patients with proliferative diabetic retinopathy (PDR) for single-cell RNA sequencing (scRNA-Seq) analyses. We also provide a detailed description of the data analysis that we implemented in a previously published study.

The quality of standard single-cell experiments often depends on the immediate processing of cells or tissues post-harvest to preserve fragile and vulnerable cell populations, unless the samples are adequately fixed and stored. Despite the recent rise in popularity of probe-based and aldehyde-fixed RNA assays, these methods face limitations in species and target availability and are not suitable for immunoprofiling or assessing chromatin accessibility. Recently, a reversible fixation strategy known as FixNCut has been successfully deployed to separate sampling from downstream applications in a reproducible and robust manner, avoiding stress or necrosis-related artifacts. In this article, we present an optimized and robust practical guide to the FixNCut protocol to aid the end-to-end adaptation of this versatile method. This protocol not only decouples tissue or cell harvesting from single-cell assays but also enables a flexible and decentralized workflow that unlocks the potential for single-cell analysis as well as unconventional study designs that were previously considered unfeasible.

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME’s intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche.

Proliferating cells need to cope with extensive cytoskeletal and nuclear remodeling as they prepare to divide. These events are tightly regulated by the nuclear translocation of the cyclin B1-CDK1 complex, that is partly dependent on nuclear tension. Standard experimental approaches do not allow the manipulation of forces acting on cells in a time-resolved manner. Here, we describe a protocol that enables dynamic mechanical manipulation of single cells with high spatial and temporal resolution and its application in the context of cell division. In addition, we also outline a method for the manipulation of substrate stiffness using polyacrylamide hydrogels. Finally, we describe a static cell confinement setup, which can be used to study the impact of prolonged mechanical stimulation in populations of cells.

Key features

• Protocol for microfabrication of confinement devices.

• Single-cell dynamic confinement coupled with high-resolution microscopy.

• Static cell confinement protocol that can be combined with super-resolution STED microscopy.

• Analysis of the mechanical control of mitotic entry in a time-resolved manner.

Graphical overview

Rapid development in single-cell chromosome conformation capture technologies has provided valuable insights into the importance of spatial genome architecture for gene regulation. However, a long-standing technical gap remains in the simultaneous characterization of three-dimensional genomes and transcriptomes in the same cell. We have described an assay named Hi-C and RNA-seq employed simultaneously (HiRES), which integrates in situ reverse transcription and chromosome conformation capture (3C) for the parallel analysis of chromatin organization and gene expression. Here, we provide a detailed implementation of the assay, using mouse embryos and cerebral cortices as examples. The versatility of this method extends beyond these two samples, with the potential to be used in various other cell types.

Key features

• A multi-omics sequencing approach to profile 3D genome structure and gene expression simultaneously in single cells.

• Compatible with animal tissues.

• One-tube amplification of both DNA and RNA components.

• Requires three days to complete.

Graphical overview

Inflammation of the gastrointestinal tract is a prevalent pathology in diseases such as inflammatory bowel disease (IBD). Currently, there are no therapies to prevent IBD, and available therapies to treat IBD are often sub-optimal. Thus, an unmet need exists to better understand the molecular mechanisms underlying intestinal tissue responses to damage and regeneration. The recent development of single-cell RNA (sc-RNA) sequencing-based techniques offers a unique opportunity to shed light on novel signaling pathways and cellular states that govern tissue adaptation or maladaptation across a broad spectrum of diseases. These approaches require the isolation of high-quality cells from tissues for downstream transcriptomic analyses. In the context of intestinal biology, there is a lack of protocols that ensure the isolation of epithelial and non-epithelial compartments simultaneously with high-quality yield. Here, we report two protocols for the isolation of epithelial and stromal cells from mouse and human colon tissues under inflammatory conditions. Specifically, we tested the feasibility of the protocols in a mouse model of dextran sodium sulfate (DSS)-induced colitis and in human biopsies from Crohn’s patients. We performed sc-RNA sequencing analysis and demonstrated that the protocol preserves most of the epithelial and stromal cell types found in the colon. Moreover, the protocol is suitable for immunofluorescence staining of surface markers for epithelial, stromal, and immune cell lineages for flow cytometry analyses. This optimized protocol will provide a new resource for scientists to study complex tissues such as the colon in the context of tissue damage and regeneration.

Key features

• This protocol allows the isolation of epithelial and stromal cells from colon tissues.

• The protocol has been optimized for tissues under inflammatory conditions with compromised cell viability.

• This protocol is suitable for experimental mouse models of colon inflammation and human biopsies.

Graphical overview

Graphical representation of the main steps for the processing of colon tissue from dextran sodium sulfate (DSS)-treated mice (upper panel) and frozen biopsies from Crohn’s patients (lower panel)