Peer Reviewed Papers Published by Nexcelom
Title & Abstract
MBAA. Brian Martyniak, Jason Bolton, Dmitry Kuksin, Suzanne M. Purseglove, Leo Li-Ying Chan
Keywords: Brettanomyces, Pseudohyphae, Image cytometry, Cellometer, Cell counting
Brettanomyces spp. have been increasingly used for brewing fermentation owing to their unique flavor and aroma. They present interesting cell morphologies composed of excessive pseudohyphae and budding, which can lead to difficulties in determining cell concentration and viability. The gold standard for cell counting is manual counting of methylene blue–stained yeasts in a hemacytometer using a standard light microscope. However, manual counting can be time consuming and has high operator-dependent variations owing to subjectivity. In recent years, the Cellometer image cytometer has been used to measure cell concentration and viability of Saccharomyces cerevisiae using acridine orange and propidium iodide viability dyes. By utilizing this method, individual cell nuclei can be directly counted in the pseudohyphae structure, which can improve the accuracy and efficiency of Brettanomyces counting, as well as eliminating the subjectivity of manual counting. In this work, a Brettanomyces propagation and fermentation experiment was conducted with the use of the Cellometer X2 image cytometer. First, the propagation experiment was performed to measure the growth, viability, and pseudohyphae percentages of B. claussenii, B. bruxellensis, and B. lambicus, as well as optimizing the software counting parameters in a controlled environment. Second, the fermentation experiment was performed to monitor and characterize the growth pattern, viability, and pseudohyphae percentages of B. lambicus and B. claussenii during beer fermentation. The proposed image cytometry method can improve the accuracy and efficiency of cell counting compared with the traditional methods, which can potentially improve the quality of beverage products employing Brettanomyces yeasts.
A rapid single cell sorting verification method using plate-based image cytometry.
Cytometry Part A. Zigon ES, Purseglove SM, Toxavidis V, Rice W, Tigges J, Chan LL.
Keywords: Celigo; cell line development; fluorescence activated cell sorting (FACS); image cytometry; monoclonality; single cell sorting
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.
Real-Time Apoptosis and Viability High-Throughput Screening of 3D Multicellular Tumor Spheroids Using the Celigo Image Cytometer
SLAS Discovery. Kessel S, Cribbes S, Bonasu S, Qiu J, Chan LL.
Keywords: 3D multicellular tumor spheroid (MCTS); Celigo image cytometry; high-throughput; kinetic spheroid apoptosis and viability; real-time
Three-dimensional tumor spheroid models have been increasingly used to investigate and characterize cancer drug compounds. Previously, the Celigo image cytometer has demonstrated its utility in a high-throughput screening manner for evaluating potential drug candidates in a 3D multicellular tumor spheroid (MCTS) primary screen. In addition, we have developed real-time kinetic caspase 3/7 apoptosis and propidium iodide viability 3D MCTS assays, both of which can be used in a secondary screen to better characterize the hit compounds. In this work, we monitored the kinetic apoptotic and cytotoxic effects of 14 compounds in 3D MCTS produced from the glioblastoma cell line U87MG in 384-well plates for 9 days. The kinetic results allowed the categorization of the effects from 14 drug compounds into early and late cytotoxic, apoptotic, cytostatic, and no effects. The real-time apoptosis and viability screening method can serve as an improved secondary screen to better understand the mechanism of action of these potential drug candidates identified from the primary screen, allowing one to identify a more qualified drug candidate and streamline the drug discovery process of research and development
Optical Coherence Tomography Detects Necrotic Regions and Volumetrically Quantifies Multicellular Tumor Spheroids
Cancer Research. Huang Y, Wang S, Guo Q, Kessel S, Rubinoff I, Chan LL, Li P, Liu Y, Qiu J, Zhou C
Keywords: Multicellular Tumor Spheroids
Three-dimensional (3D) tumor spheroid models have gained increased recognition as important tools in cancer research and anticancer drug development. However, currently available imaging approaches used in high-throughput screening drug discovery platforms, for example, bright-field, phase contrast, and fluorescence microscopies, are unable to resolve 3D structures deep inside (>50 μm) tumor spheroids. In this study, we established a label-free, noninvasive optical coherence tomography (OCT) imaging platform to characterize 3D morphologic and physiologic information of multicellular tumor spheroids (MCTS) growing from approximately 250 to 600 μm in height over 21 days. In particular, tumor spheroids of two cell lines, glioblastoma (U-87MG) and colorectal carcinoma (HCT116), exhibited distinctive evolutions in their geometric shapes at late growth stages. Volumes of MCTS were accurately quantified using a voxel-based approach without presumptions of their geometries. In contrast, conventional diameter-based volume calculations assuming perfect spherical shape resulted in large quantification errors. Furthermore, we successfully detected necrotic regions within these tumor spheroids based on increased intrinsic optical attenuation, suggesting a promising alternative of label-free viability tests in tumor spheroids. Therefore, OCT can serve as a promising imaging modality to characterize morphologic and physiologic features of MCTS, showing great potential for high-throughput drug screening.
Real-time viability and apoptosis kinetic detection method of 3D multicellular tumor spheroids using the Celigo Image Cytometer
Cytometry Part A. Kessel S, Cribbes S, Bonasu S, Rice W, Qiu J, Chan LL.
Keywords: 3D multicellular tumor spheroid; Celigo; high-throughput; image cytometry; kinetic apoptosis; kinetic viability; real-time
The development of three-dimensional (3D) multicellular tumor spheroid models for cancer drug discovery research has increased in the recent years. The use of 3D tumor spheroid models may be more representative of the complex in vivo tumor microenvironments in comparison to two-dimensional (2D) assays. Currently, viability of 3D multicellular tumor spheroids has been commonly measured on standard plate-readers using metabolic reagents such as CellTiter-Glo® for end point analysis. Alternatively, high content image cytometers have been used to measure drug effects on spheroid size and viability. Previously, we have demonstrated a novel end point drug screening method for 3D multicellular tumor spheroids using the Celigo Image Cytometer. To better characterize the cancer drug effects, it is important to also measure the kinetic cytotoxic and apoptotic effects on 3D multicellular tumor spheroids. In this work, we demonstrate the use of PI and caspase 3/7 stains to measure viability and apoptosis for 3D multicellular tumor spheroids in real-time. The method was first validated by staining different types of tumor spheroids with PI and caspase 3/7 and monitoring the fluorescent intensities for 16 and 21 days. Next, PI-stained and nonstained control tumor spheroids were digested into single cell suspension to directly measure viability in a 2D assay to determine the potential toxicity of PI. Finally, extensive data analysis was performed on correlating the time-dependent PI and caspase 3/7 fluorescent intensities to the spheroid size and necrotic core formation to determine an optimal starting time point for cancer drug testing. The ability to measure real-time viability and apoptosis is highly important for developing a proper 3D model for screening tumor spheroids, which can allow researchers to determine time-dependent drug effects that usually are not captured by end point assays. This would improve the current tumor spheroid analysis method to potentially better identify more qualified cancer drug candidates for drug discovery research.
Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer
Journal of Immunological Methods.Zhang H, Chan LL, Rice W, Kassam N, Longhi MS, Zhao H, Robson SC, Gao W, Wu Y
Keywords: Antibody discovery; Celigo; High-throughput; Hybridoma screening; Image cytometry
Hybridoma screening is a critical step for antibody discovery, which necessitates prompt identification of potential clones from hundreds to thousands of hybridoma cultures against the desired immunogen. Technical issues associated with ELISA- and flow cytometry-based screening limit accuracy and diminish high-throughput capability, increasing time and cost. Conventional ELISA screening with coated antigen is also impractical for difficult-to-express hydrophobic membrane antigens or multi-chain protein complexes. Here, we demonstrate novel high-throughput screening methodology employing the Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression. The image cytometry-based high-throughput screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on Chinese hamster ovary (CHO) cells. Next, the sensitivity of the image cytometer was demonstrated by serial dilution of purified CD39 antibody. Celigo was used to measure antibody affinities of commercial and in-house antibodies to membrane-bound CD39. This cell-based screening procedure can be completely accomplished within one day, significantly improving throughput and efficiency of hybridoma screening. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ~5ng/mL, with concurrent Kd binding affinity coefficient determination. We propose that this screening method will greatly facilitate antibody discovery and screening technologies.
Assessment of Cell Viability with Single-, Dual-, and Multi-Staining Methods Using Image Cytometry
Methods in Molecular Biology. Chan LL, McCulley KJ, Kessel SL
Keywords: Celigo; Cellometer; Enzymatic stain; Fluorescent stain; Image cytometry; Multi-stain method; Nucleic acid stain; Trypan blue; Viability
The ability to accurately measure cell viability is important for any cell-based assay. Traditionally, viability measurements have been performed using the trypan blue exclusion method on a hemacytometer, which allows researchers to visually distinguish viable from nonviable cells. While the trypan blue method can work for cell lines or primary cells that have been rigorously purified, in more complex samples such as PBMCs, bone marrow, whole blood, or any sample with low viability, this method can lead to errors. In recent years, advances in optics and fluorescent dyes have led to the development of automated benchtop image-based cell counters for rapid cell concentration and viability measurement. In this work, we demonstrate the use of image-based cytometry for cell viability detection using single-, dual-, or multi-stain techniques. Single-staining methods using nucleic acid stains such as EB, PI, 7-AAD, DAPI, SYTOX Green, and SYTOX Red, and enzymatic stains such as CFDA and Calcein AM, were performed. Dual-staining methods using AO/PI, CFDA/PI, Calcein AM/PI, Hoechst/PI, Hoechst/DRAQ7, and DRAQ5/DAPI that enumerate viable and nonviable cells were also performed. Finally, Hoechst/Calcein AM/PI was used for a multi-staining method. Fluorescent viability staining allows exclusion of cellular debris and nonnucleated cells from analysis, which can eliminate the need to perform purification steps during sample preparation and improve efficiency. Image cytometers increase speed and throughput, capture images for visual confirmation of results, and can greatly simplify cell count and viability measurements.
A novel concentration and viability detection method for Brettanomyces using the Cellometer image cytometry
Journal of Industrial Microbiology and Biotechnology Martyniak B, Bolton J, Kuksin D, Shahin SM, Chan LL.
Keywords: Brettanomyces yeast, concentration, viability, pseudohyphae, fermentation, beer, Cellometer X2, image cytometry
Brettanomyces spp. can present unique cell morphologies comprised of excessive pseudohyphae and budding, leading to difficulties in enumerating cells. The current cell counting methods include manual counting of methylene blue-stained yeasts or measuring optical densities using a spectrophotometer. However, manual counting can be time-consuming and has high operator-dependent variations due to subjectivity. Optical density measurement can also introduce uncertainties where instead of individual cells counted, an average of a cell population is measured. In contrast, by utilizing the fluorescence capability of an image cytometer to detect acridine orange and propidium iodide viability dyes, individual cell nuclei can be counted directly in the pseudohyphae chains, which can improve the accuracy and efficiency of cell counting, as well as eliminating the subjectivity from manual counting. In this work, two experiments were performed to demonstrate the capability of Cellometer image cytometer to monitor Brettanomyces concentrations, viabilities, and budding/pseudohyphae percentages. First, a yeast propagation experiment was conducted to optimize software counting parameters for monitoring the growth of Brettanomyces clausenii, Brettanomyces bruxellensis, and Brettanomyces lambicus, which showed increasing cell concentrations, and varying pseudohyphae percentages. The pseudohyphae formed during propagation were counted either as multiple nuclei or a single multi-nuclei organism, where the results of counting the yeast as a single multi-nuclei organism were directly compared to manual counting. Second, a yeast fermentation experiment was conducted to demonstrate that the proposed image cytometric analysis method can monitor the growth pattern of B. lambicus and B. clausenii during beer fermentation. The results from both experiments displayed different growth patterns, viability, and budding/pseudohyphae percentages for each Brettanomyces species. The proposed Cellometer image cytometry method can improve efficiency and eliminate operator-dependent variations of cell counting compared with the traditional methods, which can potentially improve the quality of beverage products employing Brettanomyces yeasts.
A Novel Multiparametric Drug-Scoring Method for High-Throughput Screening of 3D Multicellular Tumor Spheroids Using the Celigo Image Cytometer
SAGE Journals Scott Cribbes, Sarah Kessel, Scott McMenemy, Jean Qiu, Leo Li-Ying Chan
Keywords: 3D model, Multicellular, tumor spheroid, high-throughput screening, multiparametric, Celigo, image cytometry, Growth inhibition, Invasion, viability, apoptosis
Three-dimensional (3D) tumor models have been increasingly used to investigate and characterize cancer drug compounds. The ability to perform high-throughput screening of 3D multicellular tumor spheroids (MCTS) can highly improve the efficiency and cost-effectiveness of discovering potential cancer drug candidates. Previously, the Celigo Image Cytometer has demonstrated a novel method for high-throughput screening of 3D multicellular tumor spheroids. In this work, we employed the Celigo Image Cytometer to examine the effects of 14 cancer drug compounds on 3D MCTS of the glioblastoma cell line U87MG in 384-well plates. Using parameters such as MCTS diameter and invasion area, growth and invasion were monitored for 9 and 3 d, respectively. Furthermore, fluorescent staining with calcein AM, propidium iodide, Hoechst 33342, and caspase 3/7 was performed at day 9 posttreatment to measure viability and apoptosis. Using the kinetic and endpoint data generated, we created a novel multiparametric drug-scoring system for 3D MCTS that can be used to identify and classify potential drug candidates earlier in the drug discovery process. Furthermore, the combination of quantitative and qualitative image data can be used to delineate differences between drugs that induce cytotoxic and cytostatic effects. The 3D MCTS-based multiparametric scoring method described here can provide an alternative screening method to better qualify tested drug compounds.
A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry
Cytotechnology Leo Li-Ying Chan,Tim Smith, Kendra A. Kumph, Dmitry Kuksin, Sarah Kessel, Olivier Déry, Scott Cribbes, Ning Lai, Jean Qiu
Keywords: Image cytometry, High-throughput, Screening assay, Viability, Concentration, Acridine orange, Propidium iodide, Celigo
To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.
Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations
Analytical Biochemistry Dmitry Kuksin, Christina Arieta Kuksin, Jean Qiu, Leo Li-Ying Chan
Keywords: Calcein AM; Cellometer Vision; Flow cytometry; Image cytometry; Propidium iodide
Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flowcytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an imagecytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and imagecytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples.
High-Throughput 3D Tumor Spheroid Screening Method for Cancer Drug Discovery Using Celigo Image Cytometry
Journal of Laboratory Automation Sarah Kessel, Scott Cribbes, Olivier Déry, Dmitry Kuksin,
Keywords: 3D tumor spheroid; Celigo; glioblastoma; high-throughput screening; image cytometry; spheroid size; spheroid viability
Oncologists have investigated the effect of protein or chemical-based compounds on cancer cells to identify potential drug candidates. Traditionally, the growth inhibitory and cytotoxic effects of the drugs are first measured in 2D in vitro models, and then further tested in 3D xenograft in vivo models. Although the drug candidates can demonstrate promising inhibitory or cytotoxicity results in a 2D environment, similar effects may not be observed under a 3D environment. In this work, we developed an image-based high-throughput screening method for 3D tumor spheroids using the Celigo image cytometer. First, optimal seeding density for tumor spheroid formation was determined by investigating the cell seeding density of U87MG, a human glioblastoma cell line. Next, the dose-response effects of 17-AAG with respect to spheroid size and viability were measured to determine the IC50 value. Finally, the developed high-throughput method was used to measure the dose response of four drugs (17-AAG, paclitaxel, TMZ, and doxorubicin) with respect to the spheroid size and viability. Each experiment was performed simultaneously in the 2D model for comparison. This detection method allowed for a more efficient process to identify highly qualified drug candidates, which may reduce the overall time required to bring a drug to clinical trial.
Measuring Glycogen, Neutral Lipid, and Trehalose Contents in Yeast Using Fluorescence- Based Image Cytometry
MBAA TQ Leo Li-Ying Chan, Alexandria Kury, Alisha Wilkinson, Charlotte Berkes, and Alnoor Pirani
Keywords: Image cytometry, Viability, Vitality, Glycogen, Neutral lipid, Trehalose
The brewing, baking, and biofuel industries have long been involved with monitoring the physiological and metabolic changes of Saccharomyces cerevisiae. Breweries in particular need to observe cell health parameters such as viability, vitality, and contents of glycogen, neutral lipid, and trehalose to assess yeast cells over the course of fermentation. These physiological and metabolic variations can be qualitatively maintained via fluorescence microscopy or quantitatively evaluated via flow cytometry, using fluorescently labeled cellular markers. Conventional microscopes provide no automation for large volumes of cells, however, and although flow cytometry is capable of analyzing tens of thousands of cells, the instruments require much in the way of cost, maintenance, and training to operate. Here, we demonstrate the use of the Cellometer Vision image cytometer for the kinetic imaging and analysis of yeast cell vitality and glycogen, neutral lipid, and trehalose contents. This cytometer offers a new way for breweries of any size to monitor these characteristics throughout fermentation to produce products that are invariable and of optimal quality.
A Rapid Method for Detecting Autophagy Activity in Live Cells Using Cellometer Image Cytometry
Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging Leo Li-Ying Chan, Ning Lai, Dee Shen, Alisha R. Wilkinson, Wayne Patton, Eric Chan, Dmitry Kuksin, Bo Lin and Jean Qiu
Keywords: Cellometer Vision cytometer, Cyto-ID Green autophagy dye, fluorescence intensity, starvation and recovery assay, autophagic flux assay
Autophagy is a highly regulated and complex process that plays an important role in cellular functions such as maintaining the amino acid pool during starvation, recycling of damaged proteins and organelles, and clearing intracellular microbes. The current autophagy detection methods include fluorescence microscopy, biochemical measurement, SDS-PAGE, and Western blotting, but they are time-consuming, labor-intensive, and require much experience for accurate interpretation. Autophagy can also be analyzed on standard flow cytometers; however, they are relatively expensive and require a considerable amount of maintenance. In this work, we demonstrate a novel image cytometry method using the Cellometer image cytometer and Cyto-ID Green dye for autophagy detection in living cells. This method is validated by comparing the macroautophagic activities induced by starvation measured with image and flow cytometry. Results demonstrate similar autophagic responses, but are different in the level of fluorescence intensity changes, which may be due to the different analysis methods of the two instrument platforms. We also demonstrate the capability for drug discovery applications through the autophagy analysis of rapamycin- and tamoxifen-induced dose–response kinetics. The described image cytometry method can be a useful technique to support autophagy-based drug discovery relating to various pathological disorders.
Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry Method
Natural Killer Cells Kelsey J. McCulley and Srinivas S. Somanchi
Keywords: NK cells Cytotoxicity Image cytometry Calcein release assay Cellometer Vision
Although natural killer (NK) cells produce various cytokines that regulate other lymphocytes of the immune system, the primary effector function of NK cells is the direct cytolysis of their targets. Hence analyzing the cytotoxic potential of these lymphocytes is fundamental to understanding their biology and their clinical impact. We have previously shown that release-based cytotoxicity assays, such as calcein release assay, could potentially underestimate percent specific lysis if the entrapped reporter is not completely released and demonstrated that an Image cytometry method can overcome this caveat. In this chapter, we describe a detailed methodology to quantitate NK cell cytotoxicity using the Cellometer Vision Image Cytometry system.
Measuring Lager and Ale Yeast Viability and Vitality Using Fluorescence-Based Image Cytometry
MBAA TQ Leo Li-Ying Chan, Dan Driscoll, Dmitry Kuksin, and Stephanie Saldi
Keywords: Image cytometry, Yeast viability, Yeast vitality, Methylene blue, Hemacytometer
Saccharomyces cerevisiae has been indispensable to the production of beer for generations. Throughout the brewing process, establishing proper yeast viability and vitality is a crucial concern for proper cell growth, optimal yield, and flavor consistency. Viable yeast are those with intact membranes, whereas vital yeast are those cells with quantifiable metabolic activity and the capacity to proliferate. It is possible to have yeast that are viable but not actively propagating, and this adversely affects the fermentation process. As a rule, evaluating yeast viability involves methylene blue staining followed by manual counting with a hemacytometer. This method is time consuming and introduces human error. Here, we detail the utility of Cellometer Vision image cytometry for multichannel fluorescent assessment of yeast viability and vitality. Using nine fluorescent stains, such as nucleic acid stains (propidium iodide [PI], ethidium bromide, 4ʹ,6-diamidino-2-phenylindole, and 7-aminoactinomycin D), membrane potential, intracellular, and enzymatic dyes (oxonol, 1-anilino-8-naphthalene sulfonic acid, and carboxyfluorescein diacetate, acetoxymethyl ester [CFDA-AM]), and dual fluorescent stain combinations (acridine orange/PI and CFDA-AM/PI), we validated each against the traditional methylene blue method. Finally, Avery Brewing Company carried out a time-course analysis to compare the viability and vitality of lager and ale yeast to obtain a better understanding of the physiological and metabolic characteristics of yeast cells during the fermentation process. This information provided diagnostic criteria for monitoring the robustness of yeast in a way that may improve quality control procedures and yield beverage products that are more consistent in quality, flavor, and alcohol content.
Investigation of Macrophage Differentiation and Cytokine Production in an Undergraduate Immunology Laboratory
Journal of College Biology Teaching Charlotte Berkes and Leo Li-Ying Chan
Keywords: Immunology, image cytometry, macrophage, ELISA
We have developed a semester-long laboratory project for an undergraduate immunology course in which students study multiple aspects of macrophage biology including differentiation from progenitors in the bone marrow, activation upon stimulation with microbial ligands, expression of cell surface markers, and modulation of cytokine production. In the first part of the semester, students differentiate macrophages from mouse bone marrow stem cells and perform immunophenotyping on their macrophages using myeloid markers that are either constitutively expressed or expressed upon activation with microbial ligands. Students use a low-cost image cytometer to both visualize and quantify cellular expression of myeloid markers. Students then perform literature research, design, and execute a series of experiments aimed at investigating the role of natural anti-inflammatory compounds on TNF-α production in these macrophages. The soup-to-nuts investigative approach in which students generate “their own” macrophages ownership and accomplishment
A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry
PLoS One Somanchi SS, McCulley KJ, Somanchi A, Chan LL, Lee DA.
Keywords: NK Cell-mediated cytotoxicity, Antibody-Dependent Cell-mediated Cytotoxicity (ADCC), Chromium Release Assay, Calcein Release Assay, Image Cytometry, Direct Cell Counting, Cellometer
Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the “gold standard” for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.
JQ1 suppresses tumor growth through downregulating LDHA in ovarian cancer
Oncotarget Qiu H, Jackson AL, Kilgore JE, Zhong Y, Chan LL, Gehrig PA, Zhou C2, Bae-Jump VL.
Keywords: Ovarian cancer, JQ1, Apoptosis, Cell cycle, Mitochondrial membrane potential, Cellometer Vision, Image cytometry
Amplification and overexpression of c-Myc is commonly seen in human ovarian cancers, and this could be a potentially novel therapeutic target for this disease. JQ1, a selective small-molecule BET bromodomain (BRDs) inhibitor, has been found to suppress tumor progression in several cancer cell types. Using ovarian cancer cell lines, a transgenic mouse model, and primary cell cultures from human ovarian cancer tissues, we demonstrated that JQ1 significantly suppressed cellular proliferation and induced cell cycle arrest and apoptosis in ovarian cancer cells and mouse model via targeting c-Myc. In addition, JQ1 had multiple influences on cancer metabolism, particularly in the aerobic glycolysis pathway. JQ1 reduced both the activity and phosphorylation of LDHA, inhibited lactate production, and decreased the energy supply to ovarian cancer cell lines and tumors. Taken together, our findings suggest that JQ1 is an efficacious anti-tumor agent in ovarian cancer that is associated with cell cycle arrest, induction of apoptosis and alterations of metabolism.
Image-Based Cytometric Analysis of Fluorescent Viability and Vitality Staining Methods for Ale and Lager Fermentation Yeast
J. Am. Soc. Brew. Chem. 72(4):253-260, 2014. Stephanie Saldi, Department of Technology R&D, Nexcelom Bioscience LLC., Lawrence, MA 01843; Dan Driscoll, Avery Brewing Company, 5763 Arapahoe Ave. Ste. E, Boulder, CO 80303; and Dmitry Kuksin and Leo Li-Ying Chan, Department of Technology R&D, Nexcelom Bioscience LLC., Lawrence, MA 01843.
Keywords: Cellometer Vision, Image cytometry, Membrane integrity, Membrane potential, Methylene blue, Viability, Vitality, Yeast
Saccharomyces cerevisiae have been an essential component of beer production for centuries. The viability and vitality of yeast during a fermentation brewing process is an especially important consideration for proper cell growth, consistent flavor, and optimal production yield. The current definition of viability refers to yeast with intact membranes, while vitality refers to yeast with quantifiable metabolic activity and an ability to proliferate. Yeast may be viable, but may not be actively proliferating, which can affect the fermentation process. The traditional method for measuring yeast viability utilizes manual counting of methylene blue stained yeast cells in a hemacytometer. However, this method can be time consuming and has user-dependent variations. In this work, we demonstrate the capability of Cellometer Vision image cytometry for multi-fluorescent yeast viability and vitality measurements. Nine fluorescent stains were tested with this image cytometry system, including nucleic acid stains (PI, EB, 7-AAD, and DAPI), membrane potential, intracellular, and enzymatic stains (oxonol, MgANS, and CFDA-AM), as well as dual-fluorescent stains (AO/PI and CFDA-AM/PI). Each combination was validated against the traditional methylene blue method. Most importantly, we performed a time-course study to compare the viability and vitality of lager and ale yeast at Avery Brewing Company, which was used to better understand the physical and metabolic characteristics of yeast throughout the fermentation process. The results provide baseline knowledge for monitoring yeast health that may improve quality assurance procedures and produce more consistent beverage products (quality, flavor, alcohol content, etc.).
Evaluation of the antitumor effects of c-Myc-Max heterodimerization inhibitor 100258-F4 in ovarian cancer cells
J Transl Med. 2014 Aug 21;12(1):226. Wang J, Ma X, Jones HM, Chan L, Song F, Zhang W, Bae-Jump VL, Zhou C.
Keywords: ovarian cancer, c-Myc, 10058-F4, therapeutics, primary cell culture
Epithelial ovarian carcinoma is the most lethal gynecological cancer due to its silent onset and recurrence with resistance to chemotherapy. Overexpression of oncogene c-Myc is one of the most frequently encountered events present in ovarian carcinoma. Disrupting the function of c-Myc and its downstream target genes is a promising strategy for cancer therapy. Our objective was to evaluate the potential effects of small-molecule c-Myc inhibitor, 10058-F4, on ovarian carcinoma cells and the underlying mechanisms by which 10058-F4 exerts its actions. Using MTT assay, colony formation, flow cytometry and Annexin V FITC assays, we found that 10058-F4 significantly inhibited cell proliferation of both SKOV3 and Hey ovarian cancer cells in a dose dependent manner through induction of apoptosis and cell cycle G1 arrest. Treatment with 10058-F4 reduced cellular ATP production and ROS levels in SKOV3 and Hey cells. Consistently, primary cultures of ovarian cancer treated with 10058-F4 showed induction of caspase-3 activity and inhibition of cell proliferation in 15 of 18 cases. The response to 10058-F4 was independent the level of c-Myc protein over-expression in primary cultures of ovarian carcinoma. These novel findings suggest that the growth of ovarian cancer cells is dependent upon c-MYC activity and that targeting c-Myc-Max heterodimerization could be a potential therapeutic strategy for ovarian cancer.
Acknowledgement: A Novel Method of Inducing and Retaining Cell Cycle Synchronization in Cultures of Saccharomyces cerevisiae
Journal of the ASBC 72(2):102-109, 2014. J. Blake Layfield, Lucas R. Vann, and John D. Sheppard
Keywords: beer, cell cycle, glycerol stocks, Saccharomyces cerevisiae, seed expansion, synchronization
In conventional fermentation, at any one time, individual yeast cells are randomly distributed with respect to the stage of their growth and division cycle. The observed metabolic performance is, therefore, the result of an average of the entire population. In contrast, a synchronous population is characterized by cells that are aligned with respect to their metabolic processes, traversing the cell cycle and dividing mostly in unison. In this study, a novel method for inducing and retaining cell cycle synchronization in yeast cultures (diploid and polyploid-type) was developed using a simple and natural phased expansion method, in which the volume of the culture was increased step-wise at time periods equal to the cell doubling time. Results indicate that this method was effective in producing yeast cultures with a high degree of synchrony, verified by cell counts and fluorescent cytometry. When stored in relatively small volumes at –80°C in glycerol, the cultures maintained their synchrony upon thawing. Experiments were also conducted at the lab-scale to assess the potential use of synchronous cultures in brewing applications. The incorporation of phased seed expansion and periodic feeding of the yeast culture provided increased metabolic uniformity within the population and reduced variability in fermentation performance.
Automated Enumeration and Viability Measurement of Canine Stromal Vascular Fraction Cells Using Fluorescence-Based Image Cytometry Method.
J Fluoresc. 2014 Apr 17. Chan LL, Cohen DA, Kuksin D, Paradis BD, Qiu J.
Keywords: Stromal Vascular Fraction (SVF), Adipose-derived mesenchymal stem cell, Concentration, Viability, Acridine orange, Propidiumiodide, Image cytometry, Cellometer
In recent years, the lipoaspirate collected from adipose tissue has been seen as a valuable source of adipose-derived mesenchymal stem cells for autologous cellular therapy [1-3]. For multiple applications, adipose-derived mesenchymal stem cells are isolated from the stromal vascular fraction (SVF) of adipose tissue. Because the fresh stromal vascular fraction typically contains a heterogeneous mixture of cells [4, 5], determining cell concentration and viability is a crucial step in preparing fraction samples for downstream processing. Due to a large amount of cellular debris contained in the SVF sample, as well as counting irregularities standard manual counting can lead to inconsistent results. Advancements in imaging and optics technologies have significantly improved the image-based cytometric analysis method. In this work, we validated the use of fluorescence-based image cytometry for SVF concentration and viability measurement, by comparing to standard flow cytometry and manual hemocytometer. The concentration and viability of freshly collected canine SVF samples are analyzed, and the results highly correlated between all three methods, which validated the image cytometry method for canine SVF analysis, and potentially for SVF from other species.
Discriminating Multiplexed GFP Reporters in Primary Articular Chondrocyte Cultures Using Image Cytometry.
J Fluoresc. 2014 Apr 13. Chan LL, Huang J, Hagiwara Y, Aguila L, Rowe D.
Keywords: Articular chondrocyte, Chondrocyte differentiation, Cellometer, Image cytometry, Flow cytometry, GFP reporters, RFP reporters
Flow cytometry has become a standard tool for defining a heterogeneous cell population based on surface expressed epitopes or GFP reporters that reflect cell types or cellular differentiation. The introduction of image cytometry raised the possibility of adaptation to discriminate GFP reporters used to appreciate cell heterogeneity within the skeletal lineages. The optical filters and LEDs were optimized for the reporters used in transgenic mice expressing various fluorescent proteins. In addition, the need for compensation between eGFP and surrounding reporters due to optical cross-talk was eliminated by selecting the appropriate excitation and emission filters. Bone marrow or articular cartilage cell cultures from GFP and RFP reporter mouse lines were established to demonstrate the equivalency in functionalities of image to flow cytometry analysis. To examine the ability for monitoring primary cell differentiation, articular chondrocyte cell cultures were established from mice that were single or doubly transgenic (Dkk3eGFP and Col2A1GFPcyan), which identify the progression of superficial small articular cell to a mature chondrocyte. The instrument was able to rapidly and accurately discriminate cells that were Dkk3eGFP only, Dkk3eGFP/Col2A1GFPcyan, and Col2A1GFP, which provides a useful tool for studying the impact of culture conditions on lineage expansion and differentiation.
Morphological Observation and Analysis using Automated Image Cytometry for the Comparison of Trypan Blue and Fluorescence-based Viability Detection Method.
Cytotechnology. 2014 Mar 19. Chan LL1, Kuksin D, Laverty DJ, Saldi S, Qiu J.
Keywords: Trypan blue exclusion, Image cytometry, Cellometer, Morphology, Fluorescence, Acridine orange, Propidium iodide, Viability
The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.
Contribution of Serine, Folate and Glycine Metabolism to the ATP, NADPH and Purine Requirements of Cancer Cells.
Cell Death Dis. 2013 Oct 24, Tedeschi PM, Markert EK, Gounder M, Lin H, Dvorzhinski D, Dolfi SC, Chan LL, Qiu J, Dipaola RS, Hirshfield KM, Boros LG, Bertino JR, Oltvai ZN, Vazquez A.
Recent observations on cancer cell metabolism indicate increased serine synthesis from glucose as a marker of poor prognosis. We have predicted that a fraction of the synthesized serine is routed to a pathway for ATP production. The pathway is composed by reactions from serine synthesis, one-carbon (folate) metabolism and the glycine cleavage system (SOG pathway). Here we show that the SOG pathway is upregulated at the level of gene expression in a subset of human tumors and that its level of expression correlates with gene signatures of cell proliferation and Myc target activation. We have also estimated the SOG pathway metabolic flux in the NCI60 tumor-derived cell lines, using previously reported exchange fluxes and a personalized model of cell metabolism. We find that the estimated rates of reactions in the SOG pathway are highly correlated with the proliferation rates of these cell lines. We also observe that the SOG pathway contributes significantly to the energy requirements of biosynthesis, to the NADPH requirement for fatty acid synthesis and to the synthesis of purines. Finally, when the PC-3 prostate cancer cell line is treated with the antifolate methotrexate, we observe a decrease in the ATP levels, AMP kinase activation and a decrease in ribonucleotides and fatty acids synthesized from [1,2-(13)C2]-D-glucose as the single tracer. Taken together our results indicate that the SOG pathway activity increases with the rate of cell proliferation and it contributes to the biosynthetic requirements of purines, ATP and NADPH of cancer cells.
The Metabolic Demands of Cancer Cells are Coupled to their Size and Protein Synthesis Rates
Cancer & Metabolism. Sonia C Dolfi, Leo Li-Ying Chan, Jean Qiu, Philip M Tedeschi, Joseph R Bertino, Kim M Hirshfield, Zoltán N Oltvai and Alexei Vazquez
Keywords: Cancer metabolism, Cell size, Proliferation rate, Mesenchymal cells, Cholesterol synthesis inhibitors
Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells
JOVE. Charlotte Berkes, Leo Li-Ying Chan, Alisha Wilkinson, Benjamin Paradis
Keywords: Yeast budding , Yeast, cell cycle analysis, Image cytometry, Cellometer Vision, Acridine orange, Propidium iodide
Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.
Acknowledgement: Type I interferons induce autophagy in certain human cancer cell lines.
Autophagy. 2013 May;9(5):683-96. Schmeisser H, Fey SB, Horowitz J, Fischer ER, Balinsky CA, Miyake K, Bekisz J, Snow AL, Zoon KC.
Keywords: autophagy, human cancer cells, type I interferon, MTORC1, signal transduction, AKT, PI3K
Autophagy is an evolutionarily conserved cellular recycling mechanism that occurs at a basal level in all cells. It can be further induced by various stimuli including starvation, hypoxia, and treatment with cytokines such as IFNG/IFNγ and TGFB/TGFβ. Type I IFNs are proteins that induce an antiviral state in cells. They also have antiproliferative, proapoptotic and immunomodulatory activities. We investigated whether type I IFN can also induce autophagy in multiple human cell lines. We found that treatment with IFNA2c/IFNα2c and IFNB/IFNβ induces autophagy by 24 h in Daudi B cells, as indicated by an increase of autophagy markers MAP1LC3-II, ATG12-ATG5 complexes, and a decrease of SQSTM1 expression. An increase of MAP1LC3-II was also detected 48 h post-IFNA2c treatment in HeLa S3, MDA-MB-231, T98G and A549 cell lines. The presence of autophagosomes in selected cell lines exposed to type I IFN was confirmed by electron microscopy analysis. Increased expression of autophagy markers correlated with inhibition of MTORC1 in Daudi cells, as well as inhibition of cancer cell proliferation and changes in cell cycle progression. Concomitant blockade of either MTOR or PI3K-AKT signaling in Daudi and T98G cells treated with IFNA2c increased the level of MAP1LC3-II, indicating that the PI3K-AKT-MTORC1 signaling pathway may modulate IFN-induced autophagy in these cells. Taken together, our findings demonstrated a novel function of type I IFN as an inducer of autophagy in multiple cell lines.
Automated Quantification of Budding Saccharomyces Cerevisiae using a Novel Image Cytometry Method
J Ind Microbiol Biotechnol. 2013 Apr 5, Laverty DJ, Kury AL, Kuksin D, Pirani A, Flanagan K, Chan LL.
Keywords: Yeast budding, Yeast, cell cycle analysis, Image cytometry, Cellometer Vision, Acridine orange, Propidium iodide
The measurements of concentration, viability, and budding percentages of Saccharomyces cerevisiae are performed on a routine basis in the brewing and biofuel industries. Generation of these parameters is of great importance in a manufacturing setting, where they can aid in the estimation of product quality, quantity, and fermentation time of the manufacturing process. Specifically, budding percentages can be used to estimate the reproduction rate of yeast populations, which directly correlates with metabolism of polysaccharides and bioethanol production, and can be monitored to maximize production of bioethanol during fermentation. The traditional method involves manual counting using a hemacytometer, but this is time-consuming and prone to human error. In this study, we developed a novel automated method for the quantification of yeast budding percentages using Cellometer image cytometry. The automated method utilizes a dual-fluorescent nucleic acid dye to specifically stain live cells for imaging analysis of unique morphological characteristics of budding yeast. In addition, cell cycle analysis is performed as an alternative method for budding analysis. We were able to show comparable yeast budding percentages between manual and automated counting, as well as cell cycle analysis. The automated image cytometry method is used to analyze and characterize corn mash samples directly from fermenters during standard fermentation. Since concentration, viability, and budding percentages can be obtained simultaneously, the automated method can be integrated into the fermentation quality assurance protocol, which may improve the quality and efficiency of beer and bioethanol production processes.
Accurate Measurement of Peripheral Blood Mononuclear Cell Concentration using Image Cytometry to Eliminate RBC-induced Counting Error.
J Immunol Methods. 2013 Feb 28, Chan LL, Laverty DJ, Smith T, Nejad P, Hei H, Gandhi R, Kuksin D, Qiu J.
Keywords: Peripheral blood mononuclear cell (PBMC), Red blood cell (RBC) contamination, Image cytometry, Cellometer Vision, Acridine orange, Propidium iodide
Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. The viability and concentration of isolated PBMCs are traditionally measured by manual counting with trypan blue (TB) using a hemacytometer. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). The Cellometer image cytometry system has demonstrated the capability of automated concentration and viability detection in disposable counting chambers of unpurified mouse splenocytes and PBMCs stained with acridine orange (AO) and propidium iodide (PI) under fluorescence detection. In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods: (1) manual counting of trypan blue-stained PBMCs in hemacytometer, (2) manual counting of PBMCs in bright-field images, (3) manual counting of acetic acid lysing of RBCs with TB-stained PBMCs, (4) automated counting of acetic acid lysing of RBCs with PI-stained PBMCs, and (5) AO/PI dual staining method. The results show comparable total PBMC counting among all five methods, which validate the AO/PI staining method for PBMC measurement in the image cytometry method.
Rapid Quantification of Pathogenic Fungi by Cellometer Image-Based Cytometry
Journal of Microbiological Methods, September 2012, Berkes CA, Chan LL, Wilkinson A, Paradis B
Keywords: Image-based cytometry, Histoplasma capsulatum, Candida albicans, Cellometer Vision, Acridine orange, Propidium iodide
The objective of this study was to develop an image-based cytometric methodology for the quantification of viable pathogenic yeasts, which can offer increased sensitivity and efficiency when compared to the traditional colony forming unit (CFU) assay. Live/dead yeast quantification by flow cytometry has been previously demonstrated, however, adoption of flow cytometric detection of pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome. Here, we quantitatively assess the growth of H. capsulatum yeasts within RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with Cellometer image-based cytometry; this method faithfully recapitulates growth trends as measured by traditional CFU enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of bone marrow-derived macrophages with a GFP-expressing strain of C. albicans. To demonstrate that image-based cytometry can be used as a tool to assess the susceptibility of fungi to antifungal drugs, we perform dose response experiments with the antifungal drugs amphotericin B and itraconazole and show that image-based cytometry allows rapid assessment of the kinetics of cytotoxicity induced by these antifungals. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens, either alone or in association with host cells.
Rapid Image-based Cytometry for Comparison of Fluorescent Viability Staining Methods
Journal of fluorescence, September 2012. Chan LL, Wilkinson AR, Paradis BD, Lai N.
Keywords: Image-based cytometry (IBC), Viability, Enzymatic viability stain, Nucleic acid viability stain, Dualstaining viability method, Trypan blue exclusion method, Cellometer Vision
The ability to accurately measure cell viability is important for any cell-based research. Traditionally, viability measurements have been performed using trypan blue exclusion method on hemacytometer, which allowed researchers to visually distinguish viable from nonviable cells. However, the trypan blue method is often limited to only cell lines or primary cells that have been rigorously purified. In the recent years, small desktop image-based cell counters have been developed for rapid cell concentration and viability measurement due to advances in imaging and optics technologies as well as novel fluorescent stains. In this work, we employed the Cellometer image-based cytometer to demonstrate the ability to simplify viability detection compared to the current methods. We compared various fluorescence viability detection methods using single- or dual-staining technique. Single-staining method using nucleic acid stains including ethidium bromide, propidium iodide, 7AAD, DAPI, Sytox Green and Sytox Red, and enzymatic stains including CFDA and Calcein AM were performed. All stains produced comparable results to trypan blue exclusion method for cell line samples. Dual-staining method using AO/PI, CFDA/PI, Calcein AM/PI and Hoechst 33342/PI that enumerates viable and non-viable cells was tested on primary cell samples with high debris contents. This method allowed exclusion of cellular debris and non-nucleated cells from analysis, which can eliminate the need to perform purification step during sample preparation, and improves the efficiency of viability detection method. Overall, these image-based fluorescent cell counters can simplify assay procedures as well as capture images for visual confirmation.
A Novel Image-Based Cytometry Method for Autophagy Detection in Living Cells
Autophagy, September 2012. Leo Li-Ying Chan, Dee Shen, Alisha R. Wilkinson, Wayne Patton, Ning Lai, Eric Chan, Dmitry Kuksin, Bo Lin and Jean Qiu
Keywords: Autophagy, Autophagic flux, Chloroquine, Rapamycin, Tamoxifen, Image-based cytometry, Cellometer Vision, Cyto-ID® Green autophagy dye
Autophagy is an important cellular catabolic process that plays a variety of important roles, including maintenance of the amino acid pool during starvation, recycling of damaged proteins and organelles, and clearance of intracellular microbes. Currently employed autophagy detection methods include fluorescence microscopy, biochemical measurement, SDS-PAGE and western blotting, but they are time consuming, labor intensive, and require much experience for accurate interpretation. More recently, development of novel fluorescent probes have allowed the investigation of autophagy via standard flow cytometry. However, flow cytometers remain relatively expensive and require a considerable amount of maintenance. Previously, image-based cytometry has been shown to perform automated fluorescence-based cellular analysis comparable to flow cytometry. In this study, we developed a novel method using the Cellometer image-based cytometer in combination with Cyto-ID® Green dye for autophagy detection in live cells.
Novel Image Cytometric Method for Detection of Physiological and Metabolic Changes in Saccharomyces cerevisiae
Society for Industrial Microbiology and Biotechnology, August 2012. Chan LL, Kury A, Wilkinson A, Berkes C, Pirani A.
Keynote: Image cytometry, Cellometer Vision, Saccharomyces cerevisiae, Viability, Vitality, Glycogen, Neutral lipid, Trehalose
The studying and monitoring of physiological and metabolic changes in Saccharomyces cerevisiae (S. cerevisiae) has been a key research area for the brewing, baking, and biofuels industries, which rely on these economically important yeasts to produce their products. Specifically for breweries, physiological and metabolic parameters such as viability, vitality, glycogen, neutral lipid, and trehalose content can be measured to better understand the status of S. cerevisiae during fermentation. Traditionally, these physiological and metabolic changes can be qualitatively observed using fluorescence microscopy or flow cytometry for quantitative fluorescence analysis of fluorescently labeled cellular components associated with each parameter. However, both methods pose known challenges to the end-users. Specifically, conventional fluorescent microscopes lack automation and fluorescence analysis capabilities to quantitatively analyze large numbers of cells. Although flow cytometry is suitable for quantitative analysis of tens of thousands of fluorescently labeled cells, the instruments require a considerable amount of maintenance, highly trained technicians, and the system is relatively expensive to both purchase and maintain. In this work, we demonstrate the first use of Cellometer Vision for the kinetic detection and analysis of vitality, glycogen, neutral lipid, and trehalose content of S. cerevisiae. This method provides an important research tool for large and small breweries to study and monitor these physiological behaviors during production, which can improve fermentation conditions to produce consistent and higher-quality products.
A Novel Method for Kinetic Measurements of Rare Cell Proliferation using Cellometer Image-Based Cytometry
J Immunol Methods, March 2012. Chan LL, Zhong X, Pirani A, Lin B.
Keywords: Cell proliferation, Cellometer Vision, Image-based Cytometry (IBC), CFSE-labeling, B1 B cell, B2 B cell
Cell proliferation is an important assay for pharmaceutical and biomedical research to test the effects of a variety of treatments on cultured primary cells or cell lines. For immunological studies, the ability to perform rapid cell proliferation analysis allows the identification of potential biological reagents for inducing or inhibiting immune cell proliferation. Current cell proliferation analysis methods employ flow cytometry for fluorescence detection of CFSE-labeled cells. However, conventional flow cytometers require a considerable amount of cells per sample, which becomes an issue for kinetic measurements with rare cell population due to the lack of samples for flow cytometric analyses at multiple time points during proliferation period. Here we report the development of a novel cell proliferation kinetic detection method for low cell concentration samples using the new Cellometer Vision system.
A Rapid Detection Method for Apoptosis and Necrosis Measurement using the Cellometer Imaging Cytometry
Apoptosis, December 2011. Chan LL, Lai N, Wang E, Smith T, Yang X, Lin B.
Keywords: Imaging cytometry, Cellometer vision, Flow cytometry, Apoptosis, Necrosis, Annexin V, a-tocopheryl succinate, 2-amino-N-quinoline-8-yl benzenesulfonamide
Apoptosis and necrosis play an important role in various aspects of preclinical pharmaceutical drug discovery and validation. The ability to quickly determine the cytotoxic effect of chemical compounds on cancer cells allows researchers to efficiently identify potential drug candidates for further development in the pharmaceutical discovery pipeline. Recently, a new imaging cytometry system has been developed by Nexcelom Bioscience LLC (Lawrence, MA, USA) for fluorescence-based cell population analysis.
Cellometer Vision as an Alternative to Flow Cytometry for Cell Cycle Analysis, Mitochondrial Potential, and Immunophenotyping
Cytometry Part A, April 2011. Leo L. Chan, Xuemei Zhong, Jean Qiu, Peter Y. Li, Bo Lin
Keywords: Imaging cytometry, Cellometer Vision, Flow cytometry, Immunophenotyping, Cell cycle, Apoptosis, Mitochondrial membrane potential
Cell phenotyping and cell cycle analysis are two commonly used assays in both clinical diagnosis and biomedical research. Cell phenotyping by identifying different biomarkers is essential for the diagnosis of hematologic malignancy, sub-classifying diseases, monitoring response to treatment, predicting prognosis, detecting rare cell populations and residual malignant cells.
Rapid Detection of ABC Transporter Interaction: Potential Utility in Pharmacology
Journal of Pharmacological and Toxicological Methods, November 2010. Robey RW, Lin B, Qiu J, Chan LL, Bates SE
Keywords: ABCG2, Methods, MRP1, Fluorescence, P-glycoprotein, ABC transporter
We have developed a method to characterize the function and inhibition of ABC transporters using an automated cell counter with fluorescence detection capability. The assay was performed using stably-transfected HEK293 cells expressing P-gp, MRP1, or ABCG2 and examining transport of fluorescent substrates in the presence or absence of known inhibitors and compared to results obtained with a flow cytometer.
Direct Concentration and Viability Measurement of Yeast in Corn Mash using a Novel Imaging Cytometry Method
Journal of Industrial Microbiology Biotechnology, October 2010. Chan LL, Lyettefi EJ, Pirani A, Smith T, Qiu J, Lin B.
Keywords: Bioethanol, Corn mash, Fermentation, Yeast viability, Fluorescence detection, Cellometer, Imaging cytometry
Worldwide awareness of fossil-fuel depletion and global warming has been increasing over the last 30 years. Numerous countries, including the USA and Brazil, have introduced large-scale industrial fermentation facilities for bioethanol, biobutanol, or biodiesel production. Most of these biofuel facilities perform fermentation using standard baker’s yeasts that ferment sugar present in corn mash, sugar cane, or other glucose media. In research and development in the biofuel industry, selection of yeast strains (for higher ethanol tolerance) and fermentation conditions (yeast concentration, temperature, pH, nutrients, etc.) can be studied to optimize fermentation performance. Yeast viability measurement is needed to identify higher ethanol-tolerant yeast strains, which may prolong the fermentation cycle and increase biofuel output. In addition, yeast concentration may be optimized to improve fermentation performance. Therefore, it is important to develop a simple method for concentration and viability measurement of fermenting yeast. In this work, we demonstrate an imaging cytometry method for concentration and viability measurements of yeast in corn mash directly from operating fermenters. It employs an [Cellometer] automated cell counter, a dilution buffer, and staining solution from Nexcelom Bioscience to perform enumeration. The proposed method enables specific fluorescence detection of viable and nonviable yeasts, which can generate precise results for concentration and viability of yeast in corn mash. This method can provide an essential tool for research and development in the biofuel industry and may be incorporated into manufacturing to monitor yeast concentration and viability efficiently during the fermentation process.