Cellaca® PLX image cytometer as an alternative for immunophenotyping, GFP/RFP transfection efficiencies, and apoptosis analysis.

Cell and gene therapy is a fast-growing field for cancer therapeutics requiring reliable instrumentation and technologies. Key parameters essential for satisfying Chemistry Manufacturing and Controls criteria standards are routinely performed using flow cytometry. Recently, image cytometry was developed for cell characterization and cell-based assays but had not yet demonstrated sufficient sensitivity for surface marker detection. We developed the Cellaca® PLX image cytometry system and the respective methodologies required for immunophenotyping, GFP and RFP transfection/transduction efficiencies, and cell health analyses for routine cell characterization. All samples tested were compared directly to results from the CytoFLEX flow cytometer. PBMCs were stained with T-cell surface markers for immunophenotyping, and results show highly comparable CD3, CD4, and CD8 populations (within 5 %). GFP- or RFP-expressing cell lines were analyzed for transfection/transduction efficiencies, and the percentage positive cells and respective viabilities were equivalent on both systems. Staurosporine-treated Jurkat cells were stained for apoptotic markers, where annexin V and caspase-3 positive cells were within 5 % comparing both instruments. The proposed system may provide a complementary tool for performing routine cell-based experiments with improved efficiency and sensitivity compared to prior image cytometers, which may be significantly valuable to the cell and gene therapy field.

The Prevalence of Marine Lipophilic Phycotoxins Causes Potential Risks in a Tropical Small Island Developing State.

Tropical small island developing states (SIDS), with their geographical isolation and limited resources, heavily rely on the fisheries industry for food and revenue. The presence of marine lipophilic phycotoxins (MLPs) poses risks to their economy and human health. To understand the contamination status and potential risks, the Republic of Kiribati was selected as the representative tropical SIDS and 55 species of 256 coral reef fish encompassing multiple trophic levels and feeding strategies were collected to analyze 17 typical MLPs. Our results showed that the potential risks of ciguatoxins were the highest and approximately 62% of fish species may pose risks for consumers. Biomagnification of ciguatoxins was observed in the food web with a trophic magnification factor of 2.90. Brevetoxin-3, okadaic acid, and dinophysistoxin-1 and -2 were first reported, but the risks posed by okadaic acid and dinophysistoxins were found to be negligible. The correlation analysis revealed that fish body size and trophic position are unreliable metrics to indicate the associated risks and prevent the consumption of contaminated fish. The potential risks of MLPs in Kiribati are of concern, and our findings can serve as valuable inputs for developing food safety policies and fisheries management strategies specific to tropical SIDS contexts.

High-Throughput SARS-CoV-2 Antiviral Testing Method Using the Celigo Image Cytometer.

The COVID-19 pandemic has created a worldwide public health crisis that has since resulted in 6.8 million reported deaths. The pandemic prompted the immediate response of researchers around the world to engage in rapid vaccine development, surveillance programs, and antiviral testing, which resulted in the delivery of multiple vaccines and repurposed antiviral drug candidates. However, the emergence of new highly transmissible SARS-CoV-2 variants has renewed the desire for discovering new antiviral drug candidates with high efficacy against the emerging variants of concern. Traditional antiviral testing methods employ the plaque-reduction neutralization tests (PRNTs), plaque assays, or RT-PCR analysis, but each assay can be tedious and time-consuming, requiring 2-3 days to complete the initial antiviral assay in biologically relevant cells, and then 3-4 days to visualize and count plaques in Vero cells, or to complete cell extractions and PCR analysis. In recent years, plate-based image cytometers have demonstrated high-throughput vaccine screening methods, which can be adopted for screening potential antiviral drug candidates. In this work, we developed a high-throughput antiviral testing method employing the Celigo Image Cytometer to investigate the efficacy of antiviral drug candidates on SARS-CoV-2 infectivity using a fluorescent reporter virus and their safety by measuring the cytotoxicity effects on the healthy host cell line using fluorescent viability stains. Compared to traditional methods, the assays defined here eliminated on average 3-4 days from our standard processing time for antiviral testing. Moreover, we were able to utilize human cell lines directly that are not typically amenable to PRNT or plaque assays. The Celigo Image Cytometer can provide an efficient and robust method to rapidly identify potential antiviral drugs to effectively combat the rapidly spreading SARS-CoV-2 virus and its variants during the pandemic.

A Multiplex Assay to Simultaneously Monitor Apoptosis and Necrosis Using the Cellaca® PLX Image Cytometer.

Apoptosis is the programmed cell death pathway that is critical for maintaining homeostasis, in which cancer cells can evade to ensure survival. For pharmaceutical drug discovery, it is important to characterize and compare different cancer therapeutics (i.e., small molecules, antibody drugs, cell therapies) that can initiate the process of apoptosis, enabling the identification of potential therapeutic candidates. In this work, we developed and demonstrated a multiplex detection method for monitoring apoptosis and necrosis with Annexin V, Caspase-3, and Propidium Iodide (PI) using the Cellaca® PLX Image Cytometer (Revvity Health Sciences, Inc., Lawrence, MA). First, apoptosis was induced in Jurkat and K562 cell lines with staurosporine over the course of 24 h, where apoptosis and necrosis were assessed at 0, 1, 1.5, 2, 4, 20, and 24 h timepoints. Samples were stained with Hoechst 33342 (total dye), Annexin V-APC (early-stage apoptosis), Caspase-3 488 (late-stage apoptosis), and PI (necrosis) at each timepoint and evaluated using image cytometry. Results showed that apoptotic factors and cascades were successfully detected along the pathway from early- to late-stage apoptosis, and ultimately necrosis. A clear trend was observed analyzing apoptotic and necrotic populations during the first 1.5 h, showing differences of up to ~15% in single Annexin V+ and Caspase-3+ populations in treated Jurkat cells, however, a significant increase in double positive apoptotic/necrotic cells for Annexin V+PI+ and Capase-3+PI+ was not observed until 20 h. Upon further analysis between apoptotic populations only, Annexin V+ only populations were higher than Caspase-3+ only populations by up to ~20% between 0 and 1.5 h. Conversely, K562 cells did not exhibit a notable change in apoptotic and necrotic populations due to low sensitivity to staurosporine. The proposed image cytometric detection method may provide an effective and efficient tool for rapid and reliable simultaneous detection of early- late-stage apoptosis, and necrosis. Therefore, allowing researchers to better characterize and screen potential cancer therapeutic drug candidates for their treatment efficacy in a higher throughput manner.

Comparison of chimeric antigen receptor-T cell-mediated cytotoxicity assays with suspension tumor cells using plate-based image cytometry method.

In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. The GFP-based method demonstrated higher sensitivity in detecting CAR-T cell-mediated cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions.

Practical Characterization Strategies for Comparison, Qualification, and Selection of Cell Viability Detection Methods for Cellular Therapeutic Product Development and Manufacturing.

Cellular therapy development and manufacturing has focused on providing novel therapeutic cell-based products for various diseases. The International Organization for Standardization (ISO) has provided guidance on critical quality attributes (CQAs) that shall be considered when testing and releasing cellular therapeutic products. Cell count and viability measurements are two of the CQAs that are determined during development, manufacturing, testing, and product release. The ISO Cell Counting Standard Part 1 and 2 addressed the needs for improving the quality of cell counting results. However, there is currently no guidance on the qualification and selection of a fit-for-purpose cell viability detection method. In this work, we present strategies for the characterization and comparison of AO/PI and AO/DAPI staining methods using the heat-killed (HK) and low temperature/nutrient-deprived (LT/ND) cell death models to evaluate the comparability of cell viability measurements and identify potential causes of differences. We compared the AO/PI and AO/DAPI staining methods using HK and LT/ND-generated dead cells, investigated the staining time effects on cell viability measurements, and determined their viability linearity with different mixtures of live and dead cells. Furthermore, we validated AO/PI and AO/DAPI cell viability measurement with a long-term cell proliferation assay. Finally, we demonstrate a practical example of cell viability measurement comparison using AO/PI and AO/DAPI on antibiotic-selected transduced Jurkat and THP-1 cells to select a fit-for-purpose method for functional genomics screening. The proposed strategies may potentially enable scientists to properly characterize, compare, and select cell viability detection methods that are critical for cellular therapeutic product development and manufacturing.

A novel image-based method for simultaneous counting of Lactobacillus and Saccharomyces in mixed culture fermentation.

Mixed microorganism cultures are prevalent in the food industry. A variety of microbiological mixtures have been used in these unique fermenting processes to create distinctive flavor profiles and potential health benefits. Mixed cultures are typically not well characterized, which may be due to the lack of simple measurement tools. Image-based cytometry systems have been employed to automatically count bacteria or yeast cells. In this work, we aim to develop a novel image cytometry method to distinguish and enumerate mixed cultures of yeast and bacteria in beer products. Cellometer X2 from Nexcelom was used to count of Lactobacillus plantarum and Saccharomyces cerevisiae in mixed cultures using fluorescent dyes and size exclusion image analysis algorithm. Three experiments were performed for validation. (1) Yeast and bacteria monoculture titration, (2) mixed culture with various ratios, and (3) monitoring a Berliner Weisse mixed culture fermentation. All experiments were validated by comparing to manual counting of yeast and bacteria colony formation. They were highly comparable with ANOVA analysis showing p-value > 0.05. Overall, the novel image cytometry method was able to distinguish and count mixed cultures consistently and accurately, which may provide better characterization of mixed culture brewing applications and produce higher quality products.

Identification of Anhydrodebromoaplysiatoxin as a Dichotomic Autophagy Inhibitor.

Dysfunctional autophagy is associated with various human diseases, e.g., cancer. The discovery of small molecules modulating autophagy with therapeutic potential could be significant. To this end, we screened the ability of a series of metabolites isolated from marine microorganisms to modulate autophagy. Anhydrodebromoaplysiatoxin (ADAT), a metabolite yielded by the marine red algae Gracilaria coronopifolia, inhibited autophagosome-lysosome fusion in mammalian cells, thereby inducing the accumulation of autophagosomes. Treatment of cells with ADAT alkalinized lysosomal pH. Interestingly, ADAT also activated the mTOR/p70S6K/FoxO3a signaling pathway, likely leading to the inhibition of autophagy induction. ADAT had little effect on apoptosis. Our results suggest that ADAT is a dichotomic autophagy inhibitor that inhibits both late-stage (autophagosome-lysosome fusion) and early-stage (autophagy induction) autophagy.

Myopic macular diseases: A review.

Recent evidence has demonstrated that the global public health burden of myopia is rising rapidly. Highly myopic eyes are associated with increased frequency of eye disorders that can lead to irreversible visual impairment. With recent technological advancement in ophthalmic imaging modalities, various macular complications associated with pathologic myopia are being elucidated. The development and progression of myopic chorioretinal atrophy, myopic macular neovascularization, myopic traction maculopathy and dome-shaped macula are vision-threatening myopic macular diseases. In order to overcome the challenges in managing patients with pathologic myopia, it is important to have a complete understanding in the natural course of these myopic macular diseases. Standardising the classification criteria of pathologic myopia is essential for enhancing clinical surveillance. Personalised pharmaceutical therapy and surgical interventions will help to optimise the treatment outcomes in patients suffering from these myopic macular diseases.

Automated Assessment of Cancer Drug Efficacy On Breast Tumor Spheroids in Aggrewell™400 Plates Using Image Cytometry.

Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo® automated imaging system, we developed a method to image and individually track thousands of spheroids within the Aggrewell™400 microwell plate over time. We demonstrate the use of calcein AM and propidium iodide staining to study the effects of known anti-cancer drugs Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen. We use the image cytometry results to quantify the fluorescence of calcein AM and PI as well as spheroid size in a dose dependent manner for each of the drugs. We observe a dose-dependent reduction in spheroid size and find that it correlates well with the viability obtained from the CellTiter96® endpoint assay. The image cytometry method we demonstrate is a convenient and high-throughput drug-response assay for breast cancer spheroids under 400 µm in diameter, and may lay a foundation for investigating other three-dimensional spheroids, organoids, and tissue samples.

Characterization of New Gambierones Produced by Gambierdiscus balechii 1123M1M10.

The benthic dinoflagellate genus Gambierdiscus is the primary producer of toxins responsible for ciguatera poisoning (CP), a food intoxication endemic in tropical and subtropical areas of the world. We used high-performance liquid chromatography tandem high-resolution mass spectrometry (HPLC-HRMS) to investigate the toxin profile of Gambierdiscus balechii 1123M1M10, which was obtained from Marakei Island (2°01'N, 173°15'E), Republic of Kiribati, located in the central Pacific Ocean. Four new gambierone analogues including 12,13-dihydro-44-methylgambierone, 38-dehydroxy-12,13-dihydro-44-methylgambierone, 38-dehydroxy-44-methylgambierone, and desulfo-hydroxyl gambierone, and two known compounds, gambierone and 44-methylgambierone, were proposed by analyzing their fragmentation behaviors and pathways. Our findings provide new insights into the toxin profile of Gambierdiscus balechii 1123M1M10, which can be used as a biomarker for species identification, and lay the foundation for further toxin isolation and bioactivity studies of gambierones.

Visualising the choriocapillaris: Histology, imaging modalities and clinical research - A review.

The choriocapillaris plays a considerable role in the normal physiology of the eye as well as in various diseases. Assessing the changes in the choriocapillaris can therefore provide important information about normal ageing and pathogenesis of visual impairment, and even some systemic diseases. In vivo imaging of the choriocapillaris has evolved from non-depth resolved, dye-based angiography to advanced, high-resolution optical coherence tomography angiography (OCTA). However, the intricate microvascular networks within the choriocapillaris are still beyond the resolving limits of most OCTA instruments. Knowledge of histology, meticulous image acquisition methods, recognition of artefact and post-acquisition processing techniques are necessary for optimising OCTA choriocapillaris images. Qualitative and quantitative analyses of the choriocapillaris provide clinical information in age-related macular degeneration (AMD), diabetic retinopathy (DR), pathologic myopia and central serous chorioretinopathy (CSC). Furthermore, studies have revealed choriocapillaris changes in posterior uveitis that are correlated with treatment outcome and have important prognostic significance. In addition to retinal diseases, choriocapillaris changes have been observed in systemic vascular diseases and complications associated with pregnancy.

Characterizing the Host Coral Proteome of Platygyra carnosa Using Suspension Trapping (S-Trap).

Stony corals form the foundation of coral reefs, which are of prominent ecological and economic significance. A robust workflow for investigating the coral proteome is essential in understanding coral biology. Here we investigated different preparative workflows and characterized the proteome of Platygyra carnosa, a common stony coral of the South China Sea. We found that a combination of bead homogenization with suspension trapping (S-Trap) preparation could yield more than 2700 proteins from coral samples. Annotation using a P. carnosa transcriptome database revealed that the majority of proteins were from the coral host cells (2140, 212, and 427 proteins from host coral, dinoflagellate, and other compartments, respectively). Label-free quantification and functional annotations indicated that a high proportion were involved in protein and redox homeostasis. Furthermore, the S-Trap method achieved good reproducibility in quantitative analysis. Although yielding a low symbiont:host ratio, the method is efficient in characterizing the coral host proteomic landscape, which provides a foundation to explore the molecular basis of the responses of coral host tissues to environmental stressors.

High-Throughput Image Cytometry Detection Method for CAR-T Transduction, Cell Proliferation, and Cytotoxicity Assays.

Chimeric antigen receptor (CAR)-T cell therapy has drawn much attention due to its recent clinical success in B-cell malignancies. In general, the CAR-T cell discovery process consists of CAR identification, T-cell activation, transduction, and expansion, as well as assessment of CAR-T cytotoxicity. The current evaluation methods for the CAR-T discovery process can be time-consuming, low-throughput and requires the preparation of multiple sacrificial samples in order to produce kinetic data. In this study, we employed the use of a plate-based image cytometer to monitor anti-CAIX (carbonic anhydrase IX) G36 CAR-T generation and assess its cytotoxic potency of direct and selective killing against CAIX+ SKRC-59 human renal cell carcinoma cells. The transduction efficiency and cytotoxicity results were analyzed using image cytometry and compared directly to flow cytometry and Chromium 51 (51 Cr) release assays, showing that image cytometry was comparable against these conventional methods. Image cytometry method streamlines the assays required during the CAR-T cell discovery process by analyzing a plate of T cells from CAR-T generation to in vitro functional assays with minimum disruption. The proposed method can reduce assay time and uses less cell samples by imaging and analyze the same plate over time without the need to sacrifice any cells. The ability to monitor kinetic data can allow additional insights into the behavior and interaction between CAR-T and target tumor cells. © 2020 International Society for Advancement of Cytometry.

Establishing a reference focal plane using beads for trypan-blue-based viability measurements.

Trypan blue dye exclusion-based cell viability measurements are highly dependent upon image quality and consistency. In order to make measurements repeatable, one must be able to reliably capture images at a consistent focal plane, and with signal-to-noise ratio within appropriate limits to support proper execution of image analysis routines. Imaging chambers and imaging systems used for trypan blue analysis can be inconsistent or can drift over time, leading to a need to assure the acquisition of images prior to automated image analysis. Although cell-based autofocus techniques can be applied, the heterogeneity and complexity of the cell samples can make it difficult to assure the effectiveness, repeatability and accuracy of the routine for each measurement. Instead of auto-focusing on cells in our images, we add control beads to the images, and use them to repeatedly return to a reference focal plane. We use bead image features that have stable profiles across a wide range of focal values and exposure levels. We created a predictive model based on image quality features computed over reference datasets. Because the beads have little variation, we can determine the reference plane from bead image features computed over a single-shot image and can reproducibly return to that reference plane with each sample. The achieved accuracy (over 95%) is within the limits of the actuator repeatability. We demonstrate that a small number of beads (less than 3 beads per image) is needed to achieve this accuracy. We have also developed an open-source Graphical User Interface called Bead Benchmarking-Focus And Intensity Tool (BB-FAIT) to implement these methods for a semi-automated cell viability analyser.

It is critical for the manufacturing and release of living cell-based therapies to determine the viability, the ratio of living cells to the total number of cells (live and dead), in the therapy. Dead cells can be a safety concern for the patient, and dosing is often based on the number of living cells which are the active ingredient of the drug product. Currently, the most common approach to evaluating cell viability is based on the staining of cell samples with the trypan blue marker of cell membrane integrity: a loss in cell membrane integrity with cell death allows the dye into the cell, which can be seen using brightfield microscopy. To classify cells as live/dead, the brightness of the cells is evaluated and cells with bright centres are considered live, while those with dark centres are considered dead. Unfortunately, this approach of staining, imaging and classification is very sensitive to image acquisition settings, including image focus and brightness. This paper introduces a method to establish the required image quality for image viability analysis, providing a tool to return to image acquisition settings that will ensure image quality even when there is variability from sample to sample. In this method, polymeric beads are added to each cell sample prior to cell viability analysis. Using image processing, we extract key features from the beads in the image such as sharpness of the edges of the beads. The image features of the cells can vary significantly from sample to sample and under different cell conditions, but image features of beads have proved to be consistent across samples. We are thus able to collect reference datasets quantifying bead features over a wide range of image acquisition settings (brightness and focus), allowing us to establish a reference focal plan for image acquisition for any cell sample based on bead features. We show that with as few as three beads per image, the reference focal plane can be found from a single acquisition of beads image data over a wide range of image focuses and brightness, allowing users to consistently acquire images for cell viability that meet pre-defined quality requirements.

Optimization of UC-MSCs cold-chain storage by minimizing temperature fluctuations using an automatic cryopreservation system.

The effective long-term cryopreservation of human mesenchymal stem cells is an essential prerequisite step and represents a critical approach for their sustained supply in basic research, regenerative medicine, and tissue engineering applications. Off-the-shelf availability of human umbilical cord-derived mesenchymal stromal cells (UC-MSCs) for regenerative medicine application requires the development of nontoxic, safe, and efficient protocols for cryopreservation. In the long-term low-temperature storage process of cells, traditional manual storage has a great impact on cell activity, recovery, and function due to repeated exposure of cells to room temperature. To minimize the effect of fluctuation in ambient temperature on stored cells, we designed an automatic cryopreservation system that handles cells under controlled temperatures. In this work, UC-MSCs were utilized to investigate and compare the influence of manual and automatic cryopreservation approaches. To simulate the manual process, the UC-MSCs were transferred back and forth repeatedly (up to 400 times) between the liquid nitrogen (LN2) tank (-150 °C) and room temperature by a robotic arm. Similarly, the UC-MSCs from the same batch were collected and transferred repeatedly between two storage units by the automatic cryopreservation system, where the cells were maintained below-150 °C throughout the cold chain process. Viability, percent recovery, adherence capability, cell proliferation, and multilineage differentiation ability were assessed for UC-MSCs. Compared to the manual approach, UC-MSCs handled by the automatic system demonstrated higher viability, percent recovery, and cell proliferation, as well as improved adherence to culture plate with greater potential in multilineage differentiation after 400 temperature cycles. The described entire cold chain system may provide a powerful tool to develop safe, reliable and efficient protocols for manufacturing and banking of UC-MSCs, improving their off-the-shelf availability for regenerative medicine applications.

Wide-field Trend-based Progression Analysis of Combined Retinal Nerve Fiber Layer and Ganglion Cell Inner Plexiform Layer Thickness: A New Paradigm to Improve Glaucoma Progression Detection.

OBJECTIVE: Evaluation of glaucoma progression with OCT has been centered on the analysis of progressive retinal nerve fiber layer (RNFL) thinning over the parapapillary region and/or progressive ganglion cell inner plexiform layer (GCIPL) thinning over the macula. We investigated (1) whether combining the RNFL and GCIPL as a single layer (i.e., RNFL-GCIPL) for wide-field progression analysis outperforms wide-field progression analysis of the RNFL or the GCIPL, and (2) whether eyes with progressive RNFL-GCIPL thinning are at risk of visual field (VF) progression. DESIGN: Prospective, longitudinal study. PARTICIPANTS: A total of 440 eyes from 236 glaucoma patients; 98 eyes from 49 healthy individuals. METHODS: OCT RNFL/GCIPL/RNFL-GCIPL thickness and VF measurements were obtained at ∼4-month intervals for ≥3 years. Progressive changes of the RNFL/GCIPL/RNFL-GCIPL thicknesses were analyzed over a wide field (12×9 mm2) covering the parapapillary region and the macula with trend-based progression analysis (TPA) controlled at a false discovery rate of 5%. VF progression was determined by the Early Manifest Glaucoma Trial criteria. MAIN OUTCOME MEASURES: Proportions of eyes with progressive RNFL/GCIPL/RNFL-GCIPL thinning; hazard ratios (HRs) for development of VF progression. RESULTS: More eyes showed progressive RNFL-GCIPL thinning (127 eyes; 28.9%, 95% confidence interval [CI]: 23.9%-33.8%) than progressive RNFL thinning (74 eyes; 16.8%, 95% CI: 13.1%-20.6%) and progressive GCIPL thinning (26 eyes; 5.9%, 95% CI: 3.7%-8.1%) in the glaucoma group over the study follow-up. Progressive RNFL-GCIPL thinning was almost always detected before or simultaneously with progressive RNFL thinning or progressive GCIPL thinning. The specificity of TPA (estimated from the healthy group) for detection of progressive RNFL-GCIPL thinning, progressive RNFL thinning, and progressive GCIPL thinning was 83.7% (95% CI: 74.9%-92.4%), 94.9% (95% CI: 90.6%-99.2%), and 96.9% (95% CI: 93.5%-100.0%), respectively. Eyes with progressive RNFL-GCIPL thinning had a higher risk to develop possible (HR: 2.4, 95% CI: 1.2-5.0) or likely (HR: 4.6, 95% CI: 1.5-14.0) VF progression, with adjustment of covariates, compared with eyes without progressive RNFL-GCIPL thinning. CONCLUSIONS: Progression analysis of RNFL-GCIPL thickness reveals a significant portion of progressing eyes that neither progression analysis of RNFL thickness nor GCIPL thickness would identify. Wide-field progression analysis of RNFL-GCIPL thickness is effective to inform the risk of VF progression in glaucoma patients.

A rapid single cell sorting verification method using plate-based image cytometry.

Single cell sorting is commonly used for ensuring monoclonality and producing homogenous target cell populations. Current single cell verification methods involve manually confirming the existence of single cells or colonies in a well using a standard light microscope. However, the manual verification method is time-consuming and highly tedious, which prompts a need for an accurate and rapid detection method for verifying single cell sorting capability. Here, we demonstrate a rapid single cell sorting verification method using the Celigo Image Cytometer. Calcein AM-stained Jurkat cells and fluorescent beads are sorted into 96-well half area microplates using the MoFlo Astrios EQ. Whole well bright field and fluorescent images are acquired and analyzed using the image cytometer in less than 8 min. The proposed single cell verification detection method in multi-well microplates can allow for quick optimization of FACS instruments at flow core laboratories, as well as improvement of downstream biological assays by accurately confirming the presence of single cells in each well.

Na+ /H+ exchanger 3 blockade ameliorates type 2 diabetes mellitus via inhibition of sodium-glucose co-transporter 1-mediated glucose absorption in the small intestine.

AIM: To elucidate the role of Na+ /H+ exchanger 3 (NHE3) in sodium-glucose co-transporter 1 (SGLT1)-mediated small intestinal brush border membrane (BBM) glucose absorption and its functional implications in type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: Human jejunal samples were obtained from patients undergoing gastrectomy. 14 C-glucose absorption was measured by liquid scintillation counting. NHE3 expression was suppressed by siRNA-mediated knockdown or augmented in Caco2 cells. Glucose and insulin tolerance in db/db and m+/db mice was assessed with oral and intraperitoneal glucose tolerance tests, and an intraperitoneal insulin tolerance test. Insulin resistance and ß-cell function were assessed using homeostatic model assessment of insulin resistance and ß-cell function. RESULTS: NHE3 expression was upregulated in db/db mouse jejunal BBM and high-glucose-treated Caco2 cells. NHE3 blockade impaired SGLT1-mediated glucose absorption in human jejunum, m+/db and db/db mouse jejunums, and Caco2 cells, via serum/glucocorticoid-regulated kinase 1 (SGK1). NHE3 knockdown suppressed SGLT1-mediated glucose uptake and reduced mRNA and protein levels of SGK1 and SGLT1, which were conversely enhanced by NHE3 overexpression. Chronic S3226 treatment diminished postprandial glucose levels and ameliorated glucose intolerance in db/db mice. CONCLUSION: NHE3 is essential in the modulation of small intestinal BBM glucose absorption. Our findings provide a rationale for future possible clinical application of NHE3 for treatment of T2DM through reducing intestinal glucose uptake and counteracting postprandial hyperglycaemia.

Massively parallel nanowell-based single-cell gene expression profiling.

BACKGROUND: Technological advances have enabled transcriptome characterization of cell types at the single-cell level providing new biological insights. New methods that enable simple yet high-throughput single-cell expression profiling are highly desirable. RESULTS: Here we report a novel nanowell-based single-cell RNA sequencing system, ICELL8, which enables processing of thousands of cells per sample. The system employs a 5,184-nanowell-containing microchip to capture ~1,300 single cells and process them. Each nanowell contains preprinted oligonucleotides encoding poly-d(T), a unique well barcode, and a unique molecular identifier. The ICELL8 system uses imaging software to identify nanowells containing viable single cells and only wells with single cells are processed into sequencing libraries. Here, we report the performance and utility of ICELL8 using samples of increasing complexity from cultured cells to mouse solid tissue samples. Our assessment of the system to discriminate between mixed human and mouse cells showed that ICELL8 has a low cell multiplet rate (< 3%) and low cross-cell contamination. We characterized single-cell transcriptomes of more than a thousand cultured human and mouse cells as well as 468 mouse pancreatic islets cells. We were able to identify distinct cell types in pancreatic islets, including alpha, beta, delta and gamma cells. CONCLUSIONS: Overall, ICELL8 provides efficient and cost-effective single-cell expression profiling of thousands of cells, allowing researchers to decipher single-cell transcriptomes within complex biological samples.