Practicalities of Cell Sorting.

Cell sorting is a technique commonly used in academic and biotechnology laboratories in order to separate out cells or particles of interest from heterogeneous populations. Cell sorters use the same principles as flow cytometry analyzers, but instead of cell populations passing to the waste of the instrument, they can be collected for further studies including DNA sequencing as well as other genomic, in vitro and in vivo experiments. This chapter aims to give an overview of cell sorting, the different types of cell sorters, details on how a cell sorter works, as well as protocols that are useful when embarking on a journey with cell sorting.

A FACS-based novel isolation technique identifies heterogeneous CTCs in oral squamous cell carcinoma.

Purpose: Isolating circulating tumour cells (CTCs) from the blood is challenging due to their low abundance and heterogeneity. Limitations of conventional CTC detection methods highlight the need for improved strategies to detect and isolate CTCs. Currently, the Food and Drug Administration (FDA)-approved CellSearch™ and other RUO techniques are not available in India. Therefore, we wanted to develop a flexible CTC detection/isolation technique that addresses the limitation(s) of currently available techniques and is suitable for various downstream applications. Methods: We developed a novel, efficient, user-friendly CTC isolation strategy combining density gradient centrifugation and immuno-magnetic hematogenous cell depletion with fluorescence-activated cell sorting (FACS)-based positive selection using multiple CTC-specific cell-surface markers. For FACS, a stringent gating strategy was optimised to exclude debris and doublets by side scatter/forward scatter (SSC/FSC) discriminator, remove dead cells by 4',6-diamidino-2-phenylindole (DAPI) staining, and eliminate non-specific fluorescence using a "dump" channel. APC-labelled anti-CD45mAB was used to gate remaining hematogenous cells, while multiple epithelial markers (EpCAM, EGFR, and Pan-Cytokeratin) and an epithelial-mesenchymal transition (EMT) marker (Vimentin) labelled with fluorescein isothiocyanate (FITC) were used to sort cancer cells. The technique was initially developed by spiking Cal 27 cancer cells into the blood of healthy donors and then validated in 95 biopsy-proven oral squamous cell carcinoma (OSCC) patients. CTCs isolated from patients were reconfirmed by Giemsa staining, immuno-staining, and whole transcriptome amplification (WTA), followed by qRT-PCR. In vitro culture and RNA sequencing (RNA-Seq) were also performed to confirm their suitability for various downstream applications. Results: The mean detection efficiency for the Cal 27 tongue cancer cells spiked in the whole blood of healthy donors was 32.82% ± 12.71%. While ~75% of our patients (71/95) had detectable CTCs, the CTC positivity was independent of the TNM staging. The isolated potential cancer cells from OSCC patients were heterogeneous in size. They expressed different CTC-specific markers in various combinations as identified by qRT-PCR after WTA in different patients. Isolated CTCs were also found to be suitable for downstream applications like short-term CTC culture and RNA-Seq. Conclusion: We developed a sensitive, specific, flexible, and affordable CTC detection/isolation technique, which is scalable to larger patient cohorts, provides a snapshot of CTC heterogeneity, isolates live CTCs ready for downstream molecular analysis, and, most importantly, is suitable for developing countries.

Multiomics Characterization of a Less Invasive Microfluidic-Based Cell Sorting Technique.

Fluorescence-activated cell sorting (FACS) is a specialized technique to isolate specific cell subpopulations with a high level of recovery and accuracy. However, the cell sorting procedure can impact the viability and metabolic state of cells. Here, we performed a comparative study and evaluated the impact of traditional high-pressure charged droplet-based and microfluidic chip-based sorting on the metabolic and phosphoproteomic profile of different cell types. While microfluidic chip-based sorted cells more closely resembled the unsorted control group for most cell types tested, the droplet-based sorted cells showed significant metabolic and phosphoproteomic alterations. In particular, greater changes in redox and energy status were present in cells sorted with the droplet-based cell sorter along with larger shifts in proteostasis. 13C-isotope tracing analysis on cells recovering postsorting revealed that the sorter-induced suppression of mitochondrial TCA cycle activity recovered faster in the microfluidic chip-based sorted group. Apart from this, amino acid and lipid biosynthesis pathways were suppressed in sorted cells, with minimum impact and faster recovery in the microfluidic chip-based sorted group. These results indicate microfluidic chip-based sorting has a minimum impact on metabolism and is less disruptive compared to droplet-based sorting.

Reversible Aptamer Staining, Sorting, and Cleaning of Cells (Clean FACS) with Antidote Oligonucleotide or Nuclease Yields Fully Responsive Cells.

The ability to reverse the binding of aptamers to their target proteins has received considerable attention for developing controllable therapeutic agents. Recently, use of aptamers as reversible cell-sorting ligands has also sparked interest. Antibodies are currently utilized for isolating cells expressing a particular cell surface receptor. The inability to remove antibodies from isolated cells following sorting greatly limits their utility for many applications. Previously, we described how a particular aptamer-antidote oligonucleotide pair can isolate cells and clean them. Here, we demonstrate that this approach is generalizable; aptamers can simultaneously recognize more than one cell type during fluorescent activated cell sorting (FACS). Moreover, we describe a novel approach to reverse aptamer binding following cell sorting using a nuclease. This alternative strategy represents a cleaning approach that does not require the generation of antidote oligonucleotides for each aptamer and will greatly reduce the cost and expand the utility of Clean FACS.

The leaf idioblastome of the medicinal plant Catharanthus roseus is associated with stress resistance and alkaloid metabolism.

Catharanthus roseus leaves produce a range of monoterpenoid indole alkaloids (MIAs) that include low levels of the anticancer drugs vinblastine and vincristine. The MIA pathway displays a complex architecture spanning different subcellular and cell type localizations, and is under complex regulation. As a result, the development of strategies to increase the levels of the anticancer MIAs has remained elusive. The pathway involves mesophyll specialized idioblasts where the late unsolved biosynthetic steps are thought to occur. Here, protoplasts of C. roseus leaf idioblasts were isolated by fluorescence-activated cell sorting, and their differential alkaloid and transcriptomic profiles were characterized. This involved the assembly of an improved C. roseus transcriptome from short- and long-read data, IDIO+. It was observed that C. roseus mesophyll idioblasts possess a distinctive transcriptomic profile associated with protection against biotic and abiotic stresses, and indicative that this cell type is a carbon sink, in contrast to surrounding mesophyll cells. Moreover, it is shown that idioblasts are a hotspot of alkaloid accumulation, suggesting that their transcriptome may hold the key to the in-depth understanding of the MIA pathway and the success of strategies leading to higher levels of the anticancer drugs.

Purification of Retinal Ganglion Cells from Differentiation Through Adult via Immunopanning and Low-Pressure Flow Cytometry.

The isolation and culturing of rodent retinal ganglion cells (RGC) is a key step in studying the function and cellular response of this crucial cell type. Typical methods used for isolation of RGCs include immunopanning or magnetic bead separation with antibodies targeting RGC specific protein markers. However, in developmental research, many of the most common markers, such as Thy-1, are not expressed in early stages of development. To help study these crucial early stage RGCs, we have developed a novel method that utilizes a transgenic mouse with a GFP tag on the protein BRN3 and a low-pressure fluorescence-activated cell sorter (FACS) system.

yGPS-P: A Yeast-Based Peptidome Screen for Studying Quality Control-Associated Proteolysis.

Quality control-associated proteolysis (QCAP) is a fundamental mechanism that maintains cellular homeostasis by eliminating improperly folded proteins. In QCAP, the exposure of normally hidden cis-acting protein sequences, termed degrons, triggers misfolded protein ubiquitination, resulting in their elimination by the proteasome. To identify the landscape of QCAP degrons and learn about their unique features we have developed an unbiased screening method in yeast, termed yGPS-P, which facilitates the determination of thousands of proteome-derived sequences that enhance proteolysis. Here we describe the fundamental features of the yGPS-P method and provide a detailed protocol for its use as a tool for degron search. This includes the cloning of a synthetic peptidome library in a fluorescence-based reporter system, and data acquisition, which entails the combination of Fluorescence-Activated Cell Sorting (FACS) and Next-Generation Sequencing (NGS). We also provide guidelines for data extraction and analysis and for the application of a machine-learning algorithm that established the evolutionarily conserved amino acid preferences and secondary structure propensities of QCAP degrons.

Targeted Enrichment of Low-Abundance and Uncharacterized Taxon Members in Complex Microbial Community with Primer-Free FISH Probes Designed from Next Generation Sequencing Dataset.

Methods to obtain high-quality assembled genomic information of rare and unclassified member species in complex microbial communities remain a high priority in microbial ecology. Additionally, the supplementation of three-dimensional spatial information that highlights the morphology and spatial interaction would provide additional insights to its ecological role in the community. Fluorescent in-situ hybridization (FISH) coupling with fluorescence-activated cell sorting (FACS) is a powerful tool that enables the detection, visualization, and separation of low-abundance microbial members in samples containing complex microbial compositions. Here, we have described the workflow from designing the appropriate FISH probes from metagenomics or metatranscriptomics datasets to the preparation and treatment of samples to be used in FISH-FACS procedures.

Identifying Key Residues in Lysine Decarboxylase for Soluble Expression Using Consensus Design Soluble Mutant Screening (ConsenSing).

Although recent advances in deep learning approaches for protein engineering have enabled quick prediction of hot spot residues improving protein solubility, the predictions do not always correspond to an actual increase in solubility under experimental conditions. Therefore, developing methods that rapidly confirm the linkage between computational predictions and empirical results is essential to the success of improving protein solubility of target proteins. Here, we present a simple hybrid approach to computationally predict hot spots possibly improving protein solubility by sequence-based analysis and empirically explore valuable mutants using split GFP as a reporter system. Our approach, Consensus design Soluble Mutant Screening (ConsenSing), utilizes consensus sequence prediction to find hot spots for improvement of protein solubility and constructs a mutant library using Darwin assembly to cover all possible mutations in one pot but still keeps the library as compact as possible. This approach allowed us to identify multiple mutants of Escherichia coli lysine decarboxylase, LdcC, with substantial increases in soluble expression. Further investigation led us to pinpoint a single critical residue for the soluble expression of LdcC and unveiled its mechanism for such improvement. Our approach demonstrated that following a protein's natural evolutionary path provides insights to improve protein solubility and/or increase protein expression by a single residue mutation, which can significantly change the profile of protein solubility.

CUT & RUN to Profile Chromatin-Bound Proteins in Primary Mouse Neural Progenitor Cells.

Cleavage under targets and release using nuclease (CUT & RUN) is an innovative method to profile histone modifications and chromatin-bound proteins genome-wide. CUT & RUN offers two distinct advantages of requiring much fewer cells and providing strong signal-to-noise ratios in deep-sequencing data. Here, we describe a workflow starting from dissociation and sorting of mouse embryonic brains, CUT & RUN, and DNA library preparation to deep sequencing. With our workflow, researchers can obtain high-quality sequencing data to profile histones and chromatin-associated proteins by using as few as 100,000 neural progenitor cells (NPCs).

Fishing for Developmental Regulatory Regions: Zebrafish Tissue-Specific ATAC-seq.

Interactions between transcription factors and regulatory DNA can be described by gene regulatory networks. These networks provide a systems-level view of embryonic tissue development. Here, we describe a protocol for the isolation, identification, and experimental manipulation of tissue-specific cis-regulatory elements during zebrafish embryonic development using low-input ATAC-seq. With the methods described, genome-wide assessments of regulatory DNA in small populations of developing tissues can be identified, allowing for the construction of gene regulatory networks.

Long-term effects of early postnatal stress on Sertoli cells.

Sertoli cells are somatic cells in testis essential for spermatogenesis, that support the development, maturation, and differentiation of germ cells. Sertoli cells are metabolically highly active and physiologically regulated by external signals, particularly factors in the blood stream. In disease conditions, circulating pathological signals may affect Sertoli cells and consequentially, alter germ cells and fertility. While the effects of stress on reproductive cells have been well studied, how Sertoli cells respond to stress remains poorly characterized. We used a mouse model of early postnatal stress to assess the effects of stress on Sertoli cells. We developed an improved strategy based on intracellular stainings and obtained enriched preparations of Sertoli cells from exposed males. We show that adult Sertoli cells have impaired electron transport chain (ETC) pathways and that several components of ETC complexes particularly complex I, III, and IV are persistently affected. We identify serum as potential mediator of the effects of stress on Sertoli cells by showing that it can recapitulate ETC alterations in primary cells. These results highlight Sertoli cells as cellular targets of stress in early life that can keep a trace of exposure until adulthood.

Determination of Nucleotide Sequences within Promoter Regions Affecting Promoter Compatibility between Zymomonas mobilis and Escherichia coli.

A promoter plays a crucial role in controlling the expression of the target gene in cells, thus being one of the key biological parts for synthetic biology practices. Although significant efforts have been made to identify and characterize promoters with different strengths in various microorganisms, the compatibility of promoters within different hosts still lacks investigation. In this study, we chose the native Pgap promoter of Zymomonas mobilis to investigate nucleotide sequences within promoter regions affecting promoter compatibility between Escherichia coli and Z. mobilis. Pgap is one of the strongest promotors in Z. mobilis that has many excellent characteristics to be developed as microbial cell factories. Using EGFP as a reporter, a Z. mobilis-derived Pgap mutant library was constructed and sorted in E. coli, with candidate promoters exhibiting high fluorescence intensity collected. A total of 53 variants were finally selected and sequenced by Sanger sequencing. The sequencing results grouped these variants into 12 different Pgap variant types, among which seven types presented higher promoter strength than native Pgap in E. coli. The next-generation sequencing technique was then employed to identify key mutations within the Pgap promoter region that affect the promoter compatibility. Finally, six important sites were identified and confirmed to help increase Pgap strength in E. coli while keeping similar strength of native Pgap in Z. mobilis. Compared to native Pgap, synthetic promoters combining these sites had enhanced strength; especially, Pgap-6M combining all six sites exhibited 20-fold greater strength than native Pgap in E. coli. This study thus not only determined six important sites affecting promoter compatibility but also confirmed a series of Pgap promoter variants with strong promoter activity in both E. coli and Z. mobilis. In addition, a strategy was established in this study to investigate and determine nucleotide sequences in promoter regions affecting promoter compatibility, which can be applied in other microorganisms to help reveal universal factors affecting promoter compatibility and design promoters with desired strengths among different microbial cell factories.

Improved high-throughput screening technique to rapidly isolate Chlamydomonas transformants expressing recombinant proteins.

The single-celled eukaryotic green alga Chlamydomonas reinhardtii has long been a model system for developing genetic tools for algae, and is also considered a potential platform for the production of high-value recombinant proteins. Identifying transformants with high levels of recombinant protein expression has been a challenge in this organism, as random integration of transgenes into the nuclear genome leads to low frequency of cell lines with high gene expression. Here, we describe the design of an optimized vector for the expression of recombinant proteins in Chlamydomonas, that when transformed and screened using a dual antibiotic selection, followed by screening using fluorescence activated cell sorting (FACS), permits rapid identification and isolation of microalgal transformants with high expression of a recombinant protein. This process greatly reduces the time required for the screening process, and can produce large populations of recombinant algae transformants with between 60 and 100% of cells producing the recombinant protein of interest, in as little as 3 weeks, that can then be used for whole population sequencing or individual clone analysis. Utilizing this new vector and high-throughput screening (HTS) process resulted in an order of magnitude improvement over existing methods, which normally produced under 1% of algae transformants expressing the protein of interest. This process can be applied to other algal strains and recombinant proteins to enhance screening efficiency, thereby speeding up the discovery and development of algal-derived recombinant protein products. KEY POINTS: • A protein expression vector using double-antibiotic resistance genes was designed • Double antibiotic selection causes fewer colonies with more positive for phenotype • Coupling the new vector with FACS improves microalgal screening efficiency > 60.

A dual aurora and lim kinase inhibitor reduces glioblastoma proliferation and invasion.

High rates of recurrence and treatment resistance in the most common malignant adult brain cancer, glioblastoma (GBM), suggest that monotherapies are not sufficiently effective. Combination therapies are increasingly pursued, but the possibility of adverse drug-drug interactions may preclude clinical implementation. Developing single molecules with multiple targets is a feasible alternative strategy to identify effective and tolerable pharmacotherapies for GBM. Here, we report the development of a novel, first-in-class, dual aurora and lim kinase inhibitor termed F114. Aurora kinases and lim kinases are involved in neoplastic cell division and cell motility, respectively. Due to the importance of these cellular functions, inhibitors of aurora kinases and lim kinases are being pursued separately as anti-cancer therapies. Using in vitro and ex vivo models of GBM, we found that F114 inhibits GBM proliferation and invasion. These results establish F114 as a promising new scaffold for dual aurora/lim kinase inhibitors that may be used in future drug development efforts for GBM, and potentially other cancers.

Molecular Evolution across Mouse Spermatogenesis.

Genes involved in spermatogenesis tend to evolve rapidly, but we lack a clear understanding of how protein sequences and patterns of gene expression evolve across this complex developmental process. We used fluorescence-activated cell sorting (FACS) to generate expression data for early (meiotic) and late (postmeiotic) cell types across 13 inbred strains of mice (Mus) spanning ∼7 My of evolution. We used these comparative developmental data to investigate the evolution of lineage-specific expression, protein-coding sequences, and expression levels. We found increased lineage specificity and more rapid protein-coding and expression divergence during late spermatogenesis, suggesting that signatures of rapid testis molecular evolution are punctuated across sperm development. Despite strong overall developmental parallels in these components of molecular evolution, protein and expression divergences were only weakly correlated across genes. We detected more rapid protein evolution on the X chromosome relative to the autosomes, whereas X-linked gene expression tended to be relatively more conserved likely reflecting chromosome-specific regulatory constraints. Using allele-specific FACS expression data from crosses between four strains, we found that the relative contributions of different regulatory mechanisms also differed between cell types. Genes showing cis-regulatory changes were more common late in spermatogenesis, and tended to be associated with larger differences in expression levels and greater expression divergence between species. In contrast, genes with trans-acting changes were more common early and tended to be more conserved across species. Our findings advance understanding of gene evolution across spermatogenesis and underscore the fundamental importance of developmental context in molecular evolutionary studies.

Detection of Circulating and Tissue Myeloid-Derived Suppressor Cells (MDSC) by Flow Cytometry.

Flow cytometry allows the multiparameter analysis of heterogeneous cell populations and is an essential tool for detecting and characterizing different cell populations from peripheral blood and dissociated tissues. Myeloid-derived suppressor cells (MDSC) are a heterogeneous and plastic group of myeloid precursors with immune-suppressive capacity, which are a characteristic feature of chronic inflammation, such as cancer. The optimal measurement of MDSC levels could be used as a biomarker for clinicians for prognosis and/or management and for researchers to track and understand the role of MDSC in different pathological diseases.The criteria for defining MDSC include phenotypic surface markers, but ideally should also include the functional immunosuppressive effect on T cells, and, if possible, assessing the main biochemical and molecular features. Two major functional mechanisms to suppress T cell responses are the production of arginase-1 and reactive oxygen species (ROS) molecules. Here is presented a nine-parameter seven-color flow cytometric assay to identify and quantify MDSC from both peripheral blood mononuclear cells (PBMC) and dissociated tissue (e.g., tumor) by using fluorescence-tagged antibodies against surface markers. Also, the intracellular levels of arginase-1 and superoxide (O2-) content were performed to potentially distinguish their functional status.

Tracking de novo protein synthesis in the activated sludge microbiome using BONCAT-FACS.

In order to ensure stable performance of engineered biotechnologies that rely on mixed microbial community systems, it is important to identify process-specific microbial traits and study their in-situ activity and responses to changing environmental conditions and system operational parameters. We used BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) in combination with Fluorescence-Activated Cell Sorting (FACS) and 16S rRNA gene amplicon sequencing to identify translationally active cells in activated sludge. We found that only a subset of the activated sludge microbiome is translationally active during the aerobic treatment phase of a full-scale sequencing batch reactor designed to enhance biological phosphorus removal from municipal wastewater. Relative abundance of amplicon sequence variants was not a reliable predictor of species activity. BONCAT-positive and -negative cells revealed a broad range of population-wide and taxa-specific translational heterogeneity. BONCAT-FACS in combination with amplicon sequencing can provide new insights into the ecophysiology of highly dynamic microbiomes in activated sludge systems.

Isolation and characterization of fetal nucleated red blood cells from maternal blood as a target for single cell sequencing-based non-invasive genetic testing.

PURPOSE: Although non-invasive prenatal testing (NIPT) based on cell-free DNA (cfDNA) in maternal plasma has been prevailing worldwide, low levels of fetal DNA fraction may lead to false-negative results. Since fetal cells in maternal blood provide a pure source of fetal genomic DNA, we aimed to establish a workflow to isolate and sequence fetal nucleated red blood cells (fNRBCs) individually as a target for NIPT. METHODS: Using male-bearing pregnancy cases, we isolated fNRBCs individually from maternal blood by FACS, and obtained their genomic sequence data through PCR screening with a Y-chromosome marker and whole-genome amplification (WGA)-based whole-genome sequencing. RESULTS: The PCR and WGA efficiencies of fNRBC candidates were consistently lower than those of control cells. Sequencing data analyses revealed that although the majority of the fNRBC candidates were confirmed to be of fetal origin, many of the WGA-based genomic libraries from fNRBCs were considered to have been amplified from a portion of genomic DNA. CONCLUSIONS: We established a workflow to isolate and sequence fNRBCs individually. However, our results demonstrated that, to make cell-based NIPT targeting fNRBCs feasible, cell isolation procedures need to be further refined such that the nuclei of fNRBCs are kept intact.

Feeder-Free Differentiation Assay for Mouse Hematopoietic Stem and Progenitor Cells.

Hematopoietic stem cells (HSCs) are responsible for replenishing immune cells and reside in bone marrow (BM) niches, which provide all cellular and molecular components required for their lifelong maintenance and differentiation. Although HSCs have been extensively analyzed and characterized, their ex vivo expansion, which constitutes a promising approach for therapeutic development in regenerative medicine, remains challenging. Here, we describe an original in vitro system allowing to quantify by flow cytometry the differentiation of mouse HSCs into lineage-primed multipotent hematopoietic progenitors (MPPs) in a cytokine-supplemented feeder-free medium.