Fluorescence-activated Cell Sorting System Research Articles

Scalable Dual-Fluorescence Assay for Functional Interpretation of HNF-4α Missense Variants.

AimThe study aimed to develop a scalable dual-fluorescence assay in cells to enable the functional interpretation of HNF-4α missense variants identified in exome sequencing, which can be used to guide clinical diagnosis.MethodsUsing mOrange2 and GFP fluorescence proteins to track the expression of HNF-4α (HNF-4α-mOrange2) and reporter activity under the control of the HNF-1α promoter (pHNF1A-GFP), respectively, we designed a dual-fluorescence assay to evaluate the expression level, cellular localization, and transcriptional function of HNF-4α simultaneously in live cells. To assess the scalable characteristic of the assay, a small library containing five previously reported mutations and wild-type HNF-4α was constructed. Cells infected with this library were sorted into different populations through fluorescence-activated cell sorting (FACS) according to the transcription activity and expression abundance. Cloning and Sanger sequencing were used to detect the mutations of the different groups. High content screening (HCS) assay was used for the validation of individual mutants in the function and expression point of view.ResultsHNF-4α-mOrange2 exhibited nuclear localization and transactivation capability on the HNF-1α promoter as physical HNF-4α does. The expression of HNF-4α-mOrange2 shows a 6-fold induction of GFP expression compared to the control without HNF-4α-mOrange2, which was significantly abolished by the known loss-of-function mutant M373R. The different performances of wild-type and mutant M373R made them distinguishable in the FACS system, empowering the scalable capability of this assay for classifying large numbers of variants combining functional stratification and sequencing. Further application of the assay in the small library showed that three cell populations were seen grouped as Normal (same transactivation as wild type), Reducedexp_nor (reduced transactivation with normal or higher expression), and Reducedexp_low (reduced transactivation with lower expression). Subsequently, Sanger sequencing showed that wild-type HNF-4α was in the Normal group, two mutations (M373R and G79C) were enriched in the Reducedexp_nor group, and three mutations (C115S, L272P, and F83C) belonged to the Reducedexp_low group. These results were validated by further imaging data using HCS assay for individual mutation.ConclusionsOur study proposes a scalable and informative approach for the characterization of the variants in HNF-4α genes in a quantitative and high-throughput manner.

An integrated high-throughput microfluidic circulatory fluorescence-activated cell sorting system (μ-CFACS) for the enrichment of rare cells.

There is an increasing need for the enrichment of rare cells in the clinical environments of precision medicine, personalized medicine, and regenerative medicine. With the possibility of becoming the next-generation cell sorters, microfluidic fluorescence-activated cell sorting (μ-FACS) devices have been developed to avoid cross-contamination, minimize device footprint, and eliminate bio-aerosols. However, due to highly precise flow control, the achievable throughput of the μ-FACS system is generally lower than the throughput of conventional FACS devices. Here, we report a fully integrated high-throughput microfluidic circulatory fluorescence-activated cell sorting (μ-CFACS) system for the enrichment of clinical rare cells. A microfluidic sorting cartridge has been developed for enriching samples through a sequential sorting process, which was further realized by the integration of both fast amplified piezoelectrically actuated on-chip valves and compact pneumatic cylinders actuated on-chip valves. At an equivalent throughput of ∼8000 events per second (eps), the purity of rare fluorescent microparticles has been significantly increased from ∼0.01% to ∼27.97%. An enrichment of ∼9400-fold from 0.009% to 81.86% has also been demonstrated for isolating fluorescently labelled MCF-7 breast cancer cells from Jurkat cells at an equivalent sorting throughput of ∼6400 eps. With the advantages of high throughput and contamination-free design, the proposed integrated μ-CFACS system provides a new option for the enrichment of clinical rare cells.

Biosensor-enabled droplet microfluidic system for the rapid screening of 3-dehydroshikimic acid produced in Escherichia coli.

Genetically encoded biosensors are powerful tools used to screen metabolite-producing microbial strains. Traditionally, biosensor-based screening approaches also use fluorescence-activated cell sorting (FACS). However, these approaches are limited by the measurement of intracellular fluorescence signals in single cells, rather than the signals associated with populations comprising multiple cells. This characteristic reduces the accuracy of screening because of the variability in signal levels among individual cells. To overcome this limitation, we introduced an approach that combined biosensors with droplet microfluidics (i.e., fluorescence-activated droplet sorting, FADS) to detect labeled cells at a multi-copy level and in an independent droplet microenvironment. We used our previously reported genetically encoded biosensor, 3-dehydroshikimic acid (3-DHS), as a model with which to establish the biosensor-based FADS screening method. We then characterized and compared the effects of the sorting method on the biosensor-based screening system by subjecting the same mutant library to FACS and FADS. Notably, our developed biosensor-enabled, droplet microfluidics-based FADS screening system yielded an improved positive mutant enrichment rate and increased productivity by the best mutant, compared with the single-cell FACS system. In conclusion, the combination of a biosensor and droplet microfluidics yielded a more efficient screening method that could be applied to the biosensor-based high-throughput screening of other metabolites.

Capillary-resident EphA7+ pericytes are multipotent cells with anti-ischemic effects through capillary formation.

The presence of pericytes (PCs) with multipotency and broad distribution along capillary suggests that microvasculature plays a role not only as a duct for blood fluid transport but also as a stem cell niche that contributes to tissue maintenance and regeneration. The lack of an appropriate marker for multipotent PCs still limits our understanding of their pathophysiological roles. We identified the novel marker EphA7 to detect multipotent PCs using microarray analysis of an immortalized PC library. PCs were isolated from microvessels of mouse subcutaneous adipose tissues, then EphA7+ PCs called capillary stem cells (CapSCs) were separated from EphA7− control PCs (ctPCs) using fluorescence‐activated cell sorting system. CapSCs had highly multipotency that enabled them to differentiate into mesenchymal and neuronal lineages compared with ctPCs. CapSCs also differentiated into endothelial cells and PCs to form capillary‐like structures by themselves. Transplantation of CapSCs into ischemic tissues significantly improved blood flow recovery in hind limb ischemia mouse model due to vascular formation compared with that of ctPCs and adipose stromal cells. These data demonstrate that EphA7 identifies a subpopulation of multipotent PCs that have high angiogenesis and regenerative potency and are an attractive target for regenerative therapies.

Enhanced avermectin production by Streptomyces avermitilis ATCC 31267 using high-throughput screening aided by fluorescence-activated cell sorting.

Avermectins, produced by Streptomyces avermitilis, are important antiparasitic agents. The use of traditional microbial breeding methods for this organism has been limited by the low-throughput shake flask-based screening process. The unique growth cycle of actinomycetes makes the establishment of a reliable high-throughput screening (HTS) process difficult. To enhance the efficiency of screening strains with high yields of avermectin, a HTS process aided by fluorescence-activated cell sorting (FACS) was established. Four different spore solutions were investigated for maintaining a relatively high viability of spores. Propidium iodide (PI) and fluorescein diacetate (FDA) were used to discriminate between dead and live spores using the FACS system. Spores stained with 7-μg/mL PI and 15-μg/mL FDA at 4°C in the dark for 30min resulted in optimum sorting. Spores were treated by atmospheric and room temperature plasma (ARTP). Single live spores were sorted and sprayed into 96-well microtiter plates containing 50μL of solid agar culture medium. Solid-liquid combinatorial microculture was used for high-throughput avermectin culture. A high-titer avermectin producer (G9) was obtained from 5760 mutants after mutagenesis and HTS. Compared with the original strain, the titer was improved by 18.9% on flask culture and 20.6% on fermenter, respectively. The HTS process established in this study could easily be transferred to other similar target products produced by actinomycetes.

Fluorescence activated cell sorting via focused traveling surface acoustic waves (FTSAWs)

Fluorescence Activated Cell Sorting (FACS) is an essential technique widely used in biomedical analyses. Microfluidics, benefiting at its low power and sample consumption, has enabled miniaturization of the existing bulk FACS equipment into cost-effective and portable devices. However, these devices still have limited efficiency and biocompatibility whose actuation is based on dielectrophoresis, optical tweezers, and gas valve. Acoustophoresis, the migration of particles in acoustic field, has recently emerged as a promising method to manipulate suspended particles in microscale, thanks to the rapid response and good biocompatibility. However, limited by the wide aperture of the acoustic field, cell encapsulation in ~100s μm of droplets is one of the necessary procedures in the demonstrated acoustophoresis based FACS system to ensure sort single cell at each actuation, increasing the complexity of the system. In present work, a FACS system actuated by focused traveling surface acoustic waves has been developed. Since the aperture of acoustic field is reduced to the same order of cells by exploiting the focused acoustic waves, the cell can be sorted one at a time without encapsulation. Separation of circulating tumor cells (CTCs) from white blood cells (WBCs) was demonstrated to show good performance of the system.

IL-6/STAT3 signaling impaired induction of cancer-antigen specific T cells via down-regulation of dendritic cells in tumor microenvironment.

590 Background: Immunosuppression in tumor microenvironments is a critical issue for cancer immunotherapy. Recently, the effectiveness of immuno-check point therapy has been reported on various types of solid tumors. But, the effectiveness in colorectal cancer was poor compared to other cancers, such as melanoma and renal cell carcinoma. Correct regulation of dendritic cell (DC) function in tumors is important for inducing anti-tumor immunity. We have been demonstrated that IL-6 inhibits antigen presentation by DCs through STAT3 activation in tumor-bearing mice. In this study, we focused on the role of the IL-6/STAT3 signaling cascade in human DCs. Methods: Both IL-6 and pSTAT3 expressions in tumor sites of colorectal cancers was verified by immunohistostaining. CD11b+CD11c+DCs in cancer tissues and PBMCs were isolated by fluorescence-activated cell sorting system and investigated about the surface molecules such as HLA-DR, T cell stimulating ability, and effector gene expression levels. Moreover, we investigated influence of IL-6/STAT3 signaling in human DCs in vitro. Results: The results of IHC revealed that IL-6 was preferentially produced by cancer-associated fibroblasts and immune cells in the tissues of colorectal cancers. In addition, it was confirmed that STAT3 was activated in tumor-infiltrating immune cells. Tumor infiltrating CD11b+CD11c+ DCs highly induced IL-6 gene, down-regulated surface expression of HLA-DR, and attenuated T cell stimulating ability. In vitro experiments showed that IL-6-mediated STAT3 activation reduced surface expression of HLA-DR. COX2, cathepsin L (CTSL), and arginase activity were are involved in the IL-6-mediated down-regulation of surface expression levels of HLA-DR expression levels on DCs. Gene expressions of CTSL, ARG1 as well as IL6 in tumor infiltrating CD11b+CD11c+DCs were much higher than those of PBMCs. Conclusions: IL-6-mediated STAT3 activation inhibits functional maturation of DCs, causing suppression of anti-tumor immunity in colorectal cancer. Therefore, inhibition of the IL-6/STAT3 signaling pathway could be a promising strategy for improving immunotherapies for colorectal cancer patients.

The Efficacy of Co-Treatment with Suberoylanilide Hydroxamic Acid and Mitomycin C on Corneal Scarring after Therapeutic Keratectomy: An Animal Study

Purpose: This study was undertaken to evaluate the efficacy of co-treatment with suberoylanilide hydroxamic acid (SAHA) and mitomycin-C (MMC) on corneal fibrosis in an in-vivo model.Materials and methods: We examined the effect of co-treatment with MMC and SAHA, a potent histone deacetylase inhibitor (HDACi), on the corneal fibrosis and haze produced in rats following photorefractive keratectomy (PRK). We further examined the toxicity of this co-treatment on human corneal epithelial (HCE) cells in-vitro.Results: The combination of MMC and SAHA efficiently suppressed corneal fibrosis and haze following PRK. At the doses tested, SAHA had no inhibitory effect on human corneal epithelial cell viability, whereas MMC-treated cells showed increased apoptotic changes in Western blot testing and a fluorescence-activated cell sorting system. Co-treatment with SAHA and a low-dose of MMC minimized corneal haze after PRK and produced a significantly lower level of cytotoxic effects than treatment with MMC alone.Conclusions: Treatment with SAHA in combination with a low dose of MMC could be a novel and effective therapeutic strategy to suppress the proliferation of corneal myofibroblasts, thereby inhibiting corneal fibrosis or haze with minimal ocular surface toxicity.

B7h (ICOS-L) Maintains Tolerance at the Fetomaternal Interface

In a successful pregnancy, the semiallogeneic fetus is not rejected by the maternal immune system, which implies tolerance mechanisms protecting fetal tissues from maternal immune attack. Here we report that the ICOS-B7h costimulatory pathway plays a critical role in maintaining the equilibrium at the fetomaternal interface. Blockade of this pathway increased fetal resorption and decreased fetal survival in an allogeneic pregnancy model (CBA female × B6 male). Locally in the placenta, levels of regulatory markers such as IDO and TGF-β1 were reduced after anti-B7h monoclonal antibody treatment, whereas levels of effector cytokines (eg, IFN-γ) were significantly increased. In secondary lymphoid organs, enhanced IFN-γ and granzyme B production (predominantly by CD8(+) T cells) was observed in the anti-B7h-treated group. The deleterious effect of B7h blockade in pregnancy was maintained only in CD4 knockout mice, not in CD8 knockout mice, which suggests a role for CD8(+) Tcells in immune regulation by the ICOS-B7h pathway. In accord, regulatory CD8(+) T cells (in particular, CD8(+)CD103(+) cells) were significantly decreased after anti-B7h monoclonal antibody treatment, and adoptive transfer of this subset abrogated the deleterious effect of B7h blockade in fetomaternal tolerance. Taken together, these data support the hypothesis that B7h blockade abrogates tolerance at the fetomaternal interface by enhancing CD8(+) effector response and reducing local immunomodulation mediated by CD8(+) regulatory T cells.

Pressure Overload Induces Early Morphological Changes in the Heart

Cardiac hypertrophy, whether pathological or physiological, induces a variety of additional morphological and physiological changes in the heart, including altered contractility and hemodynamics. Events exacerbating these changes are documented during later stages of hypertrophy (usually termed pathological hypertrophy). Few studies document the morphological and physiological changes during early physiological hypertrophy. We define acute cardiac remodeling events in response to transverse aortic constriction (TAC), including temporal changes in hypertrophy, collagen deposition, capillary density, and the cell populations responsible for these changes. Cardiac hypertrophy induced by TAC in mice was detected 2 days after surgery (as measured by heart weight, myocyte width, and wall thickness) and peaked by day 7. Picrosirius staining revealed increased collagen deposition 7 days after TAC; immunostaining and flow cytometry indicated a concurrent increase in fibroblasts. The findings correlated with angiogenesis in TAC hearts; a decrease in capillary density was observed at day 2, with recovery to sham-surgery levels by day 7. Increased pericyte levels, which were observed 2 days after TAC, may mediate this angiogenic transition. Gene expression suggests a coordinated response in growth, extracellular matrix, and angiogenic factors to mediate the observed morphological changes. Our data demonstrate that morphological changes in response to cardiovascular injury occur rapidly, and the present findings allow correlation of specific events that facilitate these changes.

Histone H3 is aberrantly phosphorylated in glutamine-repeat diseases

Double-labeling immunohistochemical studies staining with anti-ubiquitin and anti-phosphoserine antibodies and application of an enzymatic dephosphorylation technique reveal neuronal inclusions and affected nuclei to be aberrantly phosphorylated in brain tissues with patients with glutamine-repeat diseases. Regional distribution of the phosphorylated nuclei in neurons correlates with the pathology. To identify the target nuclear protein, transient expression of Huntington’s disease exon 1 gene containing an expanded glutamine repeat was generated in a cell culture and nuclear inclusions were isolated with a fluorescence-activated cell sorting system. Immunoblotting studies of the aggregated nuclear proteins using anti-phosphoserine antibody demonstrate the protein of the aberrant phosphorylation as histone H3. The immunoblots of control and diseased brain tissues demonstrate that the phosphorylation of histone H3 is commonly increased in the diseased brains. Aberrant phosphorylation of histone H3 is surmised to be a shared pathological process in glutamine-repeat diseases.

Ligand binding characteristics of a glycosylphosphatidyl inositol membrane-anchored HeLa cell folate receptor epitope-related to human milk folate binding protein.

The folate receptor (FR) in HeLa cells was characterized as to ligand binding mechanism, antigenic properties and membrane anchor in order to obtain information to be used for the design of biological agents targeting FR in malignant tumors. The receptor displayed the following binding characteristics in equilibrium dialysis experiments (37 degrees C, pH 7.4) with [3H] folate: a high-affinity type of binding that exhibited positive cooperativity with a Hill coefficient > 1.0 and an upward convex Scatchard plot, a slow radioligand dissociation at pH 7.4 becoming rapid at pH 3.5 and inhibition in the presence of other folates. The molecular size of the receptor was 100 kDa on gel filtration with Triton X-100, or similar to that of high molecular weight human milk folate binding protein (FBP). The latter protein represents a 25 kDa molecule which equipped with a hydrophobic glycosylphosphatidyl inositol (GPI) membrane anchor susceptible to cleavage by phosphatidylinositol specific phospholipase C (PI-PLC) forms micelles of 1kDa size with Triton X-100. The HeLa cell FR immunoreacted with antibodies against purified human milk FBP in ELISA, and in a fluorescence activated cell sorting system, where HeLa cells exposed to increasing concentrations of antibody showed a dose-dependent response. Exposure to PI-PLC decreased the fraction of immunolabeled cells indicating a linkage of FR to cell membranes by a GPI anchor. HeLa cells incubated with radiofolate showed a continuous uptake with time, however, with a complete suppression of uptake in the presence of an excess of cold folate. Prewash of cells at acidic pH to remove endogenous folate increased the uptake. Binding and uptake of [3H] folate was increased in cells grown in a folate-deprived medium. The HeLa FR seems to be epitope related to human milk FBP.

Isolation of a gramicidin S hyperproducing strain of Bacillus brevis by use of a fluorescence activated cell sorting system.

A gramicidin S (GS) hyperproducing mutant of Bacillus brevis was isolated by using a protein-staining fluorescence dye (fluorescein isothiocyanate, FITC), and a fluorescence-activated cell sorting system (FACS). By flow cytometry (FCM) analysis after staining with FITC, higher producing cells of the wild-type had higher fluorescence signals than cells with low productivity or cells from a GS non-producing mutant. Staining with FITC did not affect the viability of cells under the conditions chosen for FCM analysis. This enabled us to recover viable cells after sorting. After wild-type cells were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, mutants with higher fluorescence than the parental strain were obtained by cell sorting. Among them, strain 18 was chosen as a GS hyperproducer; it produced 590 micrograms GS/ml compared to 350 micrograms/ml by the wild-type strain. This method has the advantage of being able to screen large numbers of cells in a short time. Furthermore, use of the fluorescence dye technique will expand the use of FACS to the improvement of other cultures that produce metabolites that do not have a specific fluorescence or strong enough fluorescence for normal cell sorting.

Automatic cell identification and enrichment in lung cancer. I. Light scatter and fluorescence parameters.

Two physical parameters were investigated to automatically recognize cells in sputum from human squamous cell carcinoma of the lung and to separate them for preparation by the Papanicolaou methods, for human interactive identification and for automated high resolution image analysis. The two parameters, 0.5-15.0 degrees forward argon-ion laser light scatter to estimate total cell size and 546 nm Acridine orange fluorescence to approximate total cell DNA content, were measured in a flow-through fluorescence activated cell sorting system. Enrichment for neoplastic cells in three cases of squamous cell carcinoma of the lung averaged 7.8-fold over the original sputum when only green fluorescence was used and 10.5-fold using green fluorescence and forward light scatter. The average enrichment for neoplastic cells was 65.6-fold relative to polymorphonuclear deenrichment.