Abstract: The study of plant biology has traditionally focused on investigations conducted at the tissue, organ, or whole plant level. However, single-cell transcriptomics has recently emerged as an important tool for plant biology, enabling researchers to uncover the expression profiles of individual cell types within a tissue. The application of this tool has revealed new insights into cell-to-cell gene expression heterogeneity and has opened new avenues for research in plant biology. A critical step in the successful application of single-cell and single-nuclei RNA-seq (scRNA-seq and snRNA-seq) is the isolation of individual cells or nuclei, respectively, from tissue to recover their transcriptional profile. A critical step during nuclei isolation for snRNA-seq studies is Fluorescent-Activated Cell Sorting (FACS). During this step, nuclei stained with DAPI (4′,6-diamidino-2-phenylindole) can be sorted and separated from cell debris and organelles. Leaf tissue presents a unique challenge due to its high content of chloroplasts, which can interfere with obtaining high-quality results. Because DAPI can also bind to the plastid genome, these organelles will be sorted as nuclei. Thus, in tissues with a high content of chloroplasts, we have a high contamination of these organelles and an overestimation of the number of nuclei. In this study, we introduce a straightforward alternative method for isolating nuclei from Zea mays leaves with reduced chloroplast contamination. By effectively removing chloroplasts during the FACS step of our protocol, using the autofluorescence from the chloroplasts, we achieved improved alignment of reads to the genome and transcriptome. Our enhanced protocol offers a valuable solution for applying snRNA-seq in tissues with a high content of chloroplasts.

Abstract: The prognosis of a plasma cell neoplasm (PCN) varies depending on the presence of genetic abnormalities. However, detecting sensitive genetic mutations poses challenges due to the heterogeneous nature of the cell population in bone marrow aspiration. The established gold standard for cell sorting is fluorescence-activated cell sorting (FACS), which is associated with lengthy processing times, substantial cell quantities, and expensive equipment. Magnetic-activated cell sorting (MACS) can be performed without the need for FACS equipment and allows for rapid sorting of many cells, making it a practical alternative. Our objective is to conduct a comparative analysis of these two sorting techniques to assess whether MACS can viably replace FACS in clinical applications. Plasma cell purity, fluorescence in situ hybridization (FISH), and next-generation sequencing analyses were performed on FACS- and MACS-sorted bone marrow samples from 31 PCN patients. The MACS-sorted samples yielded a higher percentage of plasma cells than FACS-sorted samples under microscopy (p = 0.0156) and flow cytometry (p = 0.0313). FISH performed by two methods in 10 samples showed the same results, and the proportion of abnormal cells was significantly higher in MACS than in FACS (p = 0.001). Wilcoxon matched-pairs signed rank test analysis showed that the median of differences of variant allele frequency (VAF) of two methods (VAF of MACS minus VAF of FACS) in the DNMT3A, TET2, and ASXL1 (DTA) group was - 0.006555 (p = 0.0020), while that in the non-DTA group was 0.002805 (p = 0.0019). Ten copy number variants (CNVs) were found in both FACS- and MACS-sorted samples, eight were identified only in MACS-sorted samples, and one was detected only in FACS-sorted samples. Our study demonstrates that MACS is a viable alternative for plasma cell sorting in bone marrow samples of patients with PCN.

Abstract: Hybridomas, the first method for creating monoclonal antibodies (mAbs), were reported 50 years ago. This approach, which transformed biomedical research and laid the foundation for many of the current therapeutic, diagnostic, and research reagent applications of mAbs, is still used today, despite reported low fusion yields between short-lived B cells and immortal myeloma cells. To improve hybridoma production yields and accelerate development of new mAbs, we addressed two key limitations: 1) random pairing between myeloma cells and antibody-producing cells, and 2) low efficiency of the polyethylene-glycol-mediated fusion process. We first characterized and isolated antibody-secreting cells (ASCs) from the spleen of immunized mice before cell fusion to increase the probability of successive pairing between the most suitable cell fusion partners and favor the generation of functional hybridomas. Specifically, we developed an optimized workflow combining fluorescence-activated cell sorting with antibody secretion assays, using a panel of five cell-surface markers (CD3, TACI, CD138, MHC-II, and B220) to identify a distinct ASC subset with key characteristics. Such ASCs exhibited a plasmablast phenotype with high MHC-II expression and secreted high levels of antigen (Ag)-specific antibodies in immunized mice. We then implemented a cell electrofusion procedure adapted to low cell numbers (<106 cells), in order to perform the targeted electrofusion of TACIhighCD138high sorted ASCs. This targeted approach yielded viable hybridomas in 100% of seeded culture wells compared to only 40% for the electrofusion of unsorted cells. In particular, over 60% of hybridomas generated from TACIhighCD138high sorted ASCs secreted Ag-specific mAbs, including IgGs with high Ag binding affinity (<10-9 M). These results pave the way for a high-yield mAb production method via cell fusion, with the potential to streamline hybridoma generation and thereby expand access to mAbs.

Abstract: Stem cell-based therapies have potential in the treatment of many degenerative and auto-immune diseases, yet the success of such therapies has been hampered by the heterogeneity of cultured cell populations. Traditional sorting, including fluorescence-activated sorting (FACS) and magnetic-activated sorting (MACS) are based on labeling technologies that may induce cell damage, introduce variance, or retain residual reagents into a therapeutic product. Here we introduce a label-free cell sorting system using artificial intelligence (AI) that combines microfluidic imaging and decision making based on deep learning to increase the accuracy and safety of stem cell therapy. The system uses real-time, high-throughput cell-in-flow imaging, and lightweight convolutional neural networks to classify subpopulations on the basis of morphology, texture, and deformability cues. Millisecond-scale actuation of the selective isolation of therapeutically potent subsets can be performed with intelligent automation, including mesenchymal stem cells of high immunomodulatory potential or induced pluripotent stem cell derivatives of low tumorigenic risk. The AI-assisted sorter enhances purity, viability and functional consistency compared to traditional methods, and reduces batch to batch variation. Such strategy offers a translational route to standardized, scalable, and safe stem cell commodities, with smart automation being a key facilitator of future-generation regenerative drug.

Abstract: Introduction Red pulp macrophages (RPMs) play a central role in iron recycling and immune regulation within the spleen, yet optimized methods for the isolation and characterization of pig RPM remain limited. Methods We compared two approaches for isolating RPMs from pig splenocytes: CD163 antibody- based sorting and magnetic-activated cell sorting (MACS), which leverages the natural iron content and autofluorescence of RPMs. Isolated cells were evaluated by flow cytometry for marker expression, and functional assays were performed to assess phagocytic activity and gene expression related to iron metabolism. Results Flow cytometry identified an autofluorescent population, a hallmark of RMPs, within the pig splenocytes. CD163-based method enriched RPMs to 71.8% autofluorescent cells, while the MACS- based approach achieved a higher yield of 81% autofluorescent cells without using antibodies, demonstrating greater cost-effectiveness and efficiency. Marker analysis revealed high expression of CD16 and CD163, moderate expression of CD11b, and low or undetectable levels of CD14, CD32, and CD169. Functionally, isolated RPMs demonstrated robust phagocytosis of senescent red blood cells and upregulation of genes involved in heme and iron metabolism. Discussion These findings establish an optimized, antibody-free protocol for efficient isolation of pig RPMs. The approach provides a reliable platform for studying splenic macrophage biology, iron homeostasis, and immunological research and splenic function studies.

Abstract: Cell sorting and differential motion are key processes in the life cycle of Dictyostelium discoideum (Dd) and many other organisms. Here we develop a mathematical model and investigate the processes with computer simulations. The slug stage of Dd is modeled with ellipsoidal cells of two types which have viscoelastic properties. Using the force-based model we find that when the two cell types have different strengths of motive forces and or different degrees of directionality one cell type sorts to the front of the slug. These findings are consistent with previously published results using a different model formation. When one cell type is more directed than the other it will consistently sort to the front of the slug. Likewise, but less efficiently, when one cell type exerts greater motive forces than the other it will sort to the front of the slug. The most efficient and robust cell sorting due to differential motion is when both methods are employed.

Abstract: Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBIhighMFGE8high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBIhighMFGE8high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBIhighMFGE8high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBIhighMFGE8high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBIhighMFGE8high UC-MSCs.

Abstract: The isolation of specific cell types of the brain is essential to study cell-type-specific differences in complex neurological diseases such as Alzheimer's disease. This protocol isolates oligodendrocytes, microglia, endothelial cells, astrocytes, and neurons from a single mouse brain. The process involves gentle tissue homogenization, debris removal, and sequential sorting of five distinct cell types. We validate cell purity and viability using flow cytometry and RT-qPCR. This protocol is well-suited for a range of downstream applications, including genomics, transcriptomics, and proteomics.

Abstract: This protocol describes an overall workflow for genomic analysis of sedimentary microbes. This workflow applies the F420 autofluorescence-based flow cytometric method, recently developed for the targeted sorting and sequencing of methanogenic archaea (Lambrecht et al., 2017), to investigate diverse lineages of (uncultivated) archaea in sedimentary environments. Pre-sorting steps (sample collection, cell extraction, and glycerol-TE cryopreservation) are optimized for the preservation of cell integrity and F420 autofluorescence in archaeal cells for downstream sequencing applications. Post-sorting step can be modified with different whole genome amplification (WGA) methods. See Stepanauskas et al. (2017) and Bowers et al. (2024) for example applications of different WGA methods (e.g., multiple displacement amplification, WGA-X, primary template-directed amplification).

Abstract: Abstract Efficient isolation of male gametes has enabled unprecedented advances in omics research, crucial for elucidating the molecular mechanisms governing male gametogenesis and fertilization. In this study, we developed a method for isolating generative and sperm cells from the economically important crop Solanum lycopersicum (tomato). A double fluorescent marker line was generated in the tomato variety Micro-Tom, employing mTurquoise and mScarlet-I fluorescent proteins under the control of promoters exhibiting preferential activity in generative and sperm cells, respectively. Then we developed a protocol that combines male gamete release from pollen grains and pollen tubes of this double fluorescent marker line and SYTOX Red live/dead cell stain to obtain viable cells by fluorescence-activated cell sorting. This allows the isolation of generative cells from mature pollen grains, and of sperm cells from pollen tubes after semi-in vivo growth, both in high quantity and purity. Additionally, an unexpected mScarlet-I signal in the vegetative nucleus, that persists until the sperm cells are formed, allows the sorting of vegetative nuclei. We anticipate that our novel double marker line will accelerate research into tomato male gametogenesis, thereby enhancing efforts to improve the resilience of fertilization processes to climate change.

Abstract: The phenomenon of cell sorting/segregation, by which cells organise spatially into clusters of specific cell type or function, is essential for tissue morphogenesis. This self-organization process involves an interplay between mechanical, biochemical, and cellular mechanisms that act across various spatial and temporal scales. Several mechanisms for cell sorting have been proposed; however, the physical nature of these mechanisms and how they lead to symmetric or asymmetric cell sorting remains unclear. Here, using experimental data from cocultures of genetically modified Human Embryonic Kidney (HEK293) cells and numerical simulations, we show the existence of a cell sorting mechanism based on transient increases in the persistence of motion of cells. This mechanism is activated on cells overexpressing the ephrinB1-related receptor EphB2 after their interaction with cells overexpressing ephrinB1. We show that this mechanism is sufficient to cause cell sorting, breaking the symmetry of the sorting dynamics, and show that the duration of this transient differential persistence state is optimal for enhancing sorting. Furthermore, we show that in combination with other interaction mechanisms, such as changes in direction-also known as contact inhibition of locomotion-and adhesion forces, differential persistence significantly reduces the timescale of sorting. Our findings offer insights into the behaviour of cell mixtures, the relevance of non-reciprocal interactions, and may provide insight into developmental processes and tissue patterning.

Abstract: Abstract Chimeric antigen receptor (CAR) T cell therapies have shown clinical success in cancer treatment. However, the compositions of the final products can differ substantially between patients, leading to variable treatment responses. Recent studies suggest that CAR T cells manufactured from defined T cell subsets show greater potency and persistence and improved predictability of therapeutic efficacy. Current clinical‐scale selection of T cell subsets relies on antibody‐based magnetic activated cell sorting, which is costly and results in suboptimal product purity and yield, presenting a significant challenge for clinical translation. Here, a high‐affinity CD62L aptamer and a traceless, sequential selection system are reported for the high‐yield and high‐purity isolation of CD62L⁺CD8⁺ T cells without residual selection labels. It is demonstrated that multiple aptamer‐reversal agent pairs can be integrated into a magnetic platform for multi‐parameter and high‐throughput cell sorting. CAR T cells manufactured from aptamer‐selected CD62L⁺CD8⁺ T cells, encompassing naïve and early memory CD8 + T cells, exhibit distinct phenotypic and functional advantages compared to those manufactured from bulk CD8 + T cells. This aptamer‐based approach has the potential to improve the clinical efficacy of current adoptive T cell therapies by enabling precise and scalable selection of T cell subsets, with broad applications beyond T cell subset selection.

Abstract: Detecting alterations in plasmid structures is often performed using conventional molecular biology. However, these methods are laborious and time-consuming for studying the conditions inducing these mutations, which prevent real-time access to cell heterogeneity during bioproduction. In this work, we propose combining both flow cytometry and fluorescence-activated cell sorting, integrated with mechanistic modelling to study conditions that lead to plasmid recombination using a limonene-producing microbial system as a case study. A gene encoding GFP was introduced downstream of the key enzymes involved in limonene biosynthesis to enable real-time kinetics monitoring and the identification of cell heterogeneity according to microscopic and flow cytometric analyses. Three different plasmid configurations (one correct and two incorrect) were identified through cell sorting based on subpopulations expressing different levels of GFP at 10 and 50 µM IPTG. Higher limonene production (530 mg/L) and lower subpopulation proportion carrying the incorrect plasmid (12%) were observed for 10 µM IPTG compared to 50 µM IPTG (96 mg/L limonene and more than 70% of cell population carrying the incorrect plasmid, respectively) in 100 mL production culture. We also managed to derive exploratory hypotheses regarding the plasmid recombination region using the model and successfully validated them experimentally. Additionally, the results also showed that limonene production was proportional to GFP fluorescence intensity. This correlation could serve as an alternative to using biosensors for a high-throughput screening process. The developed method enables rapid identification of plasmid recombination at single-cell level and correlates the heterogeneity with bioproduction performance. • Strategy to study plasmid recombination during bioproduction. • Different plasmid structures can be identified and monitored by flow cytometry. • Mathematical modelling suggests specific alterations in plasmid structures.

Abstract: ABSTRACT Environmental DNA (eDNA) metabarcoding typically relies on collecting and characterising a pool of mixed, fragmented DNA from environmental samples for species identification. Here, we introduce environmental metazoan cells (emCells), representing whole individual cells shed by macro‐organisms into aquatic ecosystems, and report on a method to successfully isolate and amplifying short amplicons to determine species identity. Using a custom fish probe and a novel multi‐factor fluorescence‐activated cell sorting (FACS) protocol on mesocosm water samples, we successfully enriched for target emCells, as confirmed by shifts in population density using FACS and imaging flow cytometry. Imaging flow cytometry demonstrated dual nuclear and mitochondrial staining of whole single cells, while multiplexed PCR assays (targeting both mitochondrial and nuclear DNA) confirmed the effective enrichment of fish emCells, with one‐quarter of sorted cells identified as fish. Sequences obtained from isolated emCells matched known species in the mesocosm, validating our approach. Despite efforts to exclude non‐target cells, diverse single‐cell eukaryotes were also recovered, highlighting the need for additional strategies to enrich for target emCells given the abundance and diversity of off‐target particles present in aquatic environments, which will be especially important for real‐world environments. Isolation and analysis of emCells could provide a versatile complementary approach to current eDNA methodologies by providing genomic information that normally requires direct sampling from live organisms.

Abstract: Gas vesicles (GVs) are highly promising contrast agents that have been actively investigated throughout the development of contrast agents. Unlike chemically synthesized materials, GVs offer distinct advantages in bio-related studies due to their biocompatibility, making them a safer alternative to existing agents. GVs are gas-filled structures found in microorganisms, enabling their host to remain buoyant in aqueous environments. Since GVs are composed of gas vesicle proteins, it is possible to genetically encode specific cell lines to express gas vesicles at will. However, whether these cells can be utilized in clinical trials to self-contain contrast agents has not been fully explored. In recent years, we have been investigating efficient methods to express GVs in stem cells, which could function as a repeatable contrast agent. To achieve this, we employed a drug selection technique to isolate stem cells that contain gas vesicle genes. Traditional isolation methods required fluorescence-activated cell sorting (FACS) followed by single-cell cloning, which was time-consuming and costly. These findings may guide researchers in cultivating GV-containing stem cells, potentially differentiating them into desired cell types for implantation into organs that require biomarkers.

Abstract: Cell sorting is an important fundamental process for the selection and purification of target cell types for cell analysis in the life sciences and medical fields. In particular, demand is...

Abstract: Conventional methods for generating knock-out or knock-in mammalian cell models using CRISPR-Cas9 genome editing often require tedious single-cell clone selection and expansion. In this study, we develop and optimise rapid and robust strategies to engineer homozygous fluorescent reporter knock-in cell pools with precise genome editing, circumventing clonal variability inherent to traditional approaches. To reduce false-positive cells associated with random integration, we optimise the design of donor DNA by removing the start codon of the fluorescent reporter and incorporating a self-cleaving T2A peptide system. Using fluorescence-assisted cell sorting (FACS), we efficiently identify and isolate the desired homozygous fluorescent knock-in clones, establishing stable cell pools that preserve parental cell line heterogeneity and faithfully reflect endogenous transcriptional regulation of the target gene. We evaluate the knock-in efficiency and rate of undesired random integration in the electroporation method with either a dual-plasmid system (sgRNA and donor DNA in two separate vectors) or a single-plasmid system (sgRNA and donor DNA combined in one vector). We further demonstrate that coupling our single-plasmid construct with an integrase-deficient lentivirus vector (IDLV) packaging system efficiently generates fluorescent knock-in reporter cell pools, offering flexibility between electroporation and lentivirus transduction methods. Notably, compared to the electroporation methods, the IDLV system significantly minimises random integration. Moreover, the resulting reporter cell lines are compatible with most of the available genome-wide sgRNA libraries, enabling unbiased CRISPR screens to identify key transcriptional regulators of a gene of interest. Overall, our methodologies provide a powerful genetic tool for rapid and robust generation of fluorescent reporter knock-in cell pools with precise genome editing by CRISPR-Cas9 for various research purposes.

Abstract: ABSTRACT Phagocytosis is a conserved cellular mechanism for food uptake, defense, and animal-microbe interactions in metazoans. How the discrimination and subsequent processing of different microbes in marine invertebrates is facilitated remains largely unknown. Thereto, we combined a recently developed phagocytic assay with proteomics analysis to compare the phagocytic activity of the sponge Halichondria panicea upon encounter with the native Hal 281 (i.e., H. panicea isolate) and the foreign NJ 1 (i.e., Nematostella vectensis isolate) Vibrio . The sponge cell fraction was recovered after Vibrio exposure of 30 and 60 min and used for cellular (fluorescence-activated cell sorting and microscopy) and proteomics analyses. While the number of phagocytically active cells was similar between the isolates ( P = 0.19), the distribution of vibrios over cell types differed ( P = 0.02) over time, with the tendency for accumulation of NJ 1 in choanocyte-like cells compared to a shift of Hal 281 being incorporated from choanocyte-like to archaeocyte-like cells. Initially, both vibrios elicited a proteomic response related to bacterial infection and immunity (e.g., ADAM10, RAPTOR), followed by an increase of lysosomal and endocytic proteins (e.g., NPC2) after 60 min. The attenuation of the immune response and concomitant increase of vesicular trafficking in Hal 281 after 60 min corroborates cellular observations suggesting the fast transfer of Hal 281 from choanocyte-like cells to archaeocyte-like cells, compared to an accumulation of NJ 1 in the former. Subtle but distinct differences suggest strain-specific discrimination between the two tested vibrios and may indicate a degree of immune specificity in sponges. IMPORTANCE Metazoans recognize and discriminate between different microbes. In marine invertebrates, the underlying mechanisms of microbial discrimination and immune specificity are, however, not well understood. Phagocytosis is a conserved cellular process from amoeba to humans that facilitates the ingestion and digestion of microbial cells and likely plays a role in this discrimination. To elucidate the molecular and cellular basis of this microbial discrimination, we examined the differential phagocytic processing of a native (i.e., sponge-isolated) and foreign (i.e., sea anemone-isolate) Vibrio in a marine sponge. Our findings revealed that both vibrios provoke an initial bacterial infection- and immune-related, followed by a lysosomal- and endocytic-related proteomic response. Nuanced differences in the cellular and molecular processing suggest a strain-specific discrimination between the two vibrios. This study investigates a mechanism for microbial discrimination in an early-divergent metazoan and may provide a valuable model for studying the evolution of immunity and its role in animal-microbe interactions.