Image Cytometry Method Research Articles

Novel cell-based high-throughput hybridoma screening method using the Celigo image cytometer for antibody discovery

Abstract Hybridoma screening is a highly important process that can identify potential clones from hybridoma cultures against the desired target antigen. Traditional screening methods using ELISA and flow cytometry presented technical issues such as limited accuracy, reduced high-throughput capability, increased time and cost. Here, we demonstrate a novel cell-based high-throughput screening method using 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 screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on CHO cells. Both target CHO and CFSE-stained wild type were co-cultured to simultaneously detect target antibodies and nonspecific binding, which can eliminate the need to produce a separate control sample for each hybridoma supernatant. Next, the image cytometer was used to screen 672 hybridoma supernatants to develop the screening workflow. The Celigo was used to quickly image and analyze antibody binding using Hoechst, Alexa Fluor 594 and CFSE fluorescence to identify high, medium, and low binding hits. In addition, the Celigo was used to measure the binding affinities of the target antibodies to membrane-bound CD39. 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 ~5 ng/mL. We propose that this method will greatly improve screening technologies and facilitate more efficient antibody discovery.

Abstract 5929: A novel image cytometric analysis method for T-cell-mediated cytotoxicity of 3D tumor spheroids

Abstract Cell-mediated cytotoxicity assays have been frequently performed to characterize cancer cytotoxic potential of immune cells, antibodies, and drug compounds. Traditionally, these assays are performed using release assays such as Cr51 (radioactivity), Calcein (fluorescence), or LDH (enzymatic). However, release assays have limitations such as the handling of hazardous material, the indirect measurement of cell death leading to an under estimation of cytotoxicity, the requirement for a large volume of cell sample, and the inability to visually confirm, image ad track the assay kinetically. In the recent years, Celigo image cytometry has been used to perform high-throughput cell-mediated cytotoxicity assays using a direct cell counting method where cancer cells (Target) are stained with Calcein AM. Upon the addition of effector immune cells with the fluorescently stained Target cells in the presence or absence of antibody or drug compounds, the Celigo image cytometer is used to capture bright-field and fluorescent images and analyze the change in Target cell count over time to determine the cytotoxicity percentage. In general, these assays are performed in a 2D cultures. The 2D assays for cancer drug discovery have had some difficulty in identifying more qualified drug candidates for clinical testing, thus there has been an increase in interest of performing cytotoxicity assays in 3D tumor models. Traditional 3D spheroid analysis methods require the use of standard microscopy, which is time-consuming and subjective. Celigo imaging cytometer has also been used to rapidly analyze drug effects on 3D tumor spheroids. In this work, we demonstrate a novel method of analyzing T cell-mediated cytotoxicity on 3D tumor spheroids in the presence of absence of ImmTAC molecules, which can promote higher T cell killing. In this experiment, MDA-MB-453 GFP expressing breast cancer cells are used to form tumor spheroids in an ultra-low attachment plate. The spheroids are then treated with primary T cells at 1:10 and 1:50 E:T ratios, as well as 10, 1, 0.1, 0.01, and 0.001 nM ImmTAC. The results showed a dose response effect of T cell killing with the addition of ImmTAC molecules by measuring spheroid size in GFP fluorescence and viability using propidium iodide. The image cytometry method was able to monitor changes in spheroid size and fluorescence over time and rapidly quantify T cell killing, which was as high as 75%. The captured bright-field and fluorescent images also clearly showed the cytotoxicity effect from the combination of T cells and ImmTAC. The ability to screen cytotoxic effects of immune cells, antibody, and drug compounds on 3D tumor spheroids can provide an alternative tumor model for identifying more qualified cancer drug candidates for drug discovery campaigns. Citation Format: Leo Li-Ying Chan, Laure Humbert, Scott Cribbes. A novel image cytometric analysis method for T-cell-mediated cytotoxicity of 3D tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5929. doi:10.1158/1538-7445.AM2017-5929

56 - A high-throughput image cytometry-based screening method for the detection of IL2-induced peripheral blood mononuclear cell-mediated cytotoxicity

Cell-mediated cytotoxicity assays have been an important functional test for investigating the cytotoxic effect of immune effector on target cancer cells. Cytotoxicity assays have traditionally been performed using the 51Chromium (51Cr) release assay, which involves labeling the tumor cells (target) with radioisotopes. When the target cells are lysed by the immune cells (effector), they release the entrapped radioactive 51Chromium, which is measured to determine the level cytotoxicity induced by the effector cells. The 51Chromium release assay is highly hazardous, time-consuming, and can only acquire end point readout. Furthermore, it can generate inconsistent results due to batch variation, ability of the target cell for 51Cr uptake, and low sensitivity. In this work, we demonstrate a novel high-throughput cytotoxicity screening assay using the Celigo imaging-cytometry method. First, live K562 target cells were stained with Calcein AM, and were co-cultured with PBMCs from two healthy donors in a 96-well plate in the presence or absence of IL-2. Direct cell counting of Calcein positive cells was used to calculate the changes in number of live target cells in the same well from T = 0 to T = 4 hours. Thus, accurate cell-mediated killing was determined by normalizing results based on time = 0. The donor PBMCs were added to each well with Calcein-stained target cells at different Effector-to-Target (E:T) ratios. The 96-well microplate was then scanned and analyzed using the Celigo image cytometer at different time points to measure the % lysis of target cell. Although it could be argued that release of a dye may not actually represent cytotoxicity by effector cells, nonetheless much like 51Cr release assay percent cytotoxicity was directly proportionate to the duration of the assay, E:T ratio used, and amount of activating cytokine present in the media, all as expected. Further, when PBMCs from both donors showed near equal level of cytotoxicity at 4 hr; upon calculation of the NK cell lytic unit, it was revealed that one donor possessed much higher level of effector function than the other . The proposed image cytometry method can scan and analyze the entire well area of the entire 96-well plate in 7 min, which can be utilized to perform high-throughput screening of potential antibody- or chemical-based cancer drug, which could be a more efficient method for academic, industry, and clinical research.

Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer

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.

Validation of image cytometry for sperm concentration measurement: Comparison with manual counting of 4010 human semen samples

Sperm concentration is an essential parameter in the diagnostic evaluation of men from infertile couples. It is usually determined by manual counting using a hemocytometer, and is therefore both laborious and subjective. We have earlier shown that a newly developed image cytometry (IC) method may be used to determine sperm concentration. Here we present a validation of the IC method by analysis of 4010 semen samples. There was high agreement between IC and manual counting at sperm concentrations above 3mill/ml and in samples with concentrations above 12mill/ml the two methods can be used interchangeable. However, we found substantial differences in samples below 3mill/ml. We also assessed the accuracy of the two methods by repeated measurements of 248 samples, which revealed that IC measurements seemed more accurate. Moreover, based on ten samples counted by several operators the IC method had a lower coefficient of variation than the manual method (5% vs 10%), indicating a better precision of the IC method.In conclusion, measurement of sperm concentration by IC can be used at concentrations above 3mill/ml and seems more accurate and precise than manual counting, making it an attractive option in the daily clinical practice.

Quantitative image cytometry for analyzing intracellular trafficking of G protein-coupled receptors on a chemical-trapping single cell array.

G protein-coupled receptors (GPCRs) are important targets in medical and pharmaceutical research fields, because they play key roles in a variety of biological processes. Recently, intracellular trafficking of GPCRs involving endosomal internalization and recycling to the plasma membrane has been studied as a regulation mechanism for GPCR activities. However, the absence of a quantitative single-cell analysis method has hampered conditional GPCR trafficking studies and the possibility of gaining significant insights into the mechanism of regulation of GPCR signaling. Here, we report a facile image cytometry method to analyze the trafficking of GPCRs. In this method, GPCR-expressing cells were arrayed with a photo-responsive cell-immobilizing reagent in a single-cell manner, and the tagged GPCR was visualized by pulse-labeling with a fluorescent dye through sortase-mediated peptide-tag ligation. We quantified the intracellular distribution changes of a pH-dependent GPCR, G2A, by time-course observation under mildly acidic and slightly basic pH conditions. The difference in pH-dependent G2A trafficking between individual cells was automatically detected by an image analysis custom software program, and simultaneously, the average distribution ratios were also determined for understanding the properties of G2A. The present method should be applicable for investigating the dynamic intracellular trafficking of a wide variety of GPCRs under various conditions in a high-throughput manner.

A novel concentration and viability detection method for Brettanomyces using the Cellometer 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.

Imaging Cytometry of Human Leukocytes with Third Harmonic Generation Microscopy.

Based on third-harmonic-generation (THG) microscopy and a k-means clustering algorithm, we developed a label-free imaging cytometry method to differentiate and determine the types of human leukocytes. According to the size and average intensity of cells in THG images, in a two-dimensional scatter plot, the neutrophils, monocytes, and lymphocytes in peripheral blood samples from healthy volunteers were clustered into three differentiable groups. Using these features in THG images, we could count the number of each of the three leukocyte types both in vitro and in vivo. The THG imaging-based counting results agreed well with conventional blood count results. In the future, we believe that the combination of this THG microscopy-based imaging cytometry approach with advanced texture analysis of sub-cellular features can differentiate and count more types of blood cells with smaller quantities of blood.

A high-throughput image cytometry-based screening method for the detection of IL2-induced peripheral blood mononuclear cell-mediated cytotoxicity

Abstract Cell-mediated cytotoxicity assays have been an important functional test for investigating the cytotoxic effect of immune effector on target cancer cells. Cytotoxicity assays have traditionally been performed using the 51Chromium (51Cr) release assay, which involves labeling the tumor cells (target) with radioisotopes. The 51Chromium release assay is highly hazardous, time-consuming, and can only acquire end point readout. Furthermore, it can generate inconsistent results due to batch variation, ability of the target cell for 51Cr uptake, and low sensitivity. In this work, we demonstrate a novel high-throughput cytotoxicity screening assay using the Celigo imaging-cytometry method. First the live K562 target cells were stained with Calcein AM, and were co-cultured with PBMCs from two healthy donors in a flat- bottom 96-well plate in the presence or absence of IL-2. Direct cell counting of Calcein positive cells was implied to calculate the changes in number of live target cells in the same well from T = 0 to T = 4 hours. Thus, accurate cell-mediated killing was determined using the “self-referencing” calculation, which could be a more direct method for assessing cytotoxicity than 51Cr release. The donor PBMCs were added to each well with different Calcein-stained target cells at Effector-to-Target (E:T) ratios. The 96-well microplate was scanned and analyzed using the Celigo image cytometer at different time points to measure the % lysis of target cell. The proposed image cytometry method can scan and analyze in 7 min, which can be utilized to perform high-throughput screening of potential antibody- or chemical-based cancer drug, which could be a more efficient method for academic, industry, and clinical research.

Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry Method.

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.

Abstract 314: A rapid 3D tumor spheroid analysis method using the Celigo imaging cytometry

Abstract The current 2D methods for cancer drug discovery have had some difficulty in identifying potential drug candidates that can be used for clinical testing. To overcome this challenge, there has been an increase in research of 3D tissue culture that facilitated the development of new in-vitro tumor model assays. Traditional 2D and 3D analysis method relied heavily on visual observation using microscopy. However, the method is time-consuming and has high variations. Automated plate-based imaging cytometer can be employed to rapidly analyze and characterize 3D tumor spheroids, which can be used to generate both quantitative and qualitative results. In this work, we demonstrate a novel 3D tumor spheroid analysis method using the Celigo imaging cytometer for spheroid counting, size analysis, tumor migration and invasion, tumor viability, and dose response of drug induced/inhibited tumor growth. The plate-based imaging cytometer utilizes bright-field and three fluorescence channels (Blue, Green, and Red) for multi-channel analysis. By utilizing the F theta lens technology, uniform bright-field image is captured for more accurate counting of the entire well. In addition, Celigo analysis software is used to report numerous parameters allowing detailed spheroid characterization. In addition to direct spheroid counting in the well, the use of specific fluorescent dyes and probes allow the user to define viable and hypoxic areas within spheroids and monitor migration and invasion on or into supporting cells and/or tissues. The results showed that Celigo imaging cytometer can accurately count and measure spheroid sizes in response to drug induction. Furthermore, tumor migration and invasion were clearly observed and quantified in the captured images. By utilizing the 3D spheroid imaging cytometry method, researchers can quickly characterize and quantify tumor spheroids, which can improve the efficiency of identification of potential cancer drug candidates. Citation Format: Leo Li-Ying Chan, Scott Cribbes, Maria Vinci, Sarah Kessel, Lisa Patterson, Sue Eccles. A rapid 3D tumor spheroid analysis method using the Celigo imaging cytometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 314. doi:10.1158/1538-7445.AM2015-314

Abstract 1303: Quantification of natural killer cell-mediated cytotoxicity using Celigo imaging cytometry

Abstract Cytotoxicity assays play a central role in studying the function of immune effector cells such as cytolytic T lymphocytes (CTL) and natural killer (NK) cells. Traditionally, cytotoxicity assays have been performed using 51Chromium (51Cr) and Calcein release assays. The assays involve labeling tumor cells (target) with radioisotope or fluorescent dyes, when the target cells are subjected to cytolysis by CTLs or NK cells (effector), they releases the entrapped labels into the media upon lysis. The amount of labels in the media is measured to determine the level of cytotoxicity the effectors have induced. These traditional methods may generate inconsistent results due to low sensitivity caused by poor loading efficiency and high spontaneous release of the reagents. In this work, we demonstrate a novel cytotoxicity assay using the Celigo imaging cytometry method. Utilizing imaging cytometry, direct cell counting of live fluorescent target cells can be performed, which is a direct method for assessment of cytotoxicity. Human NK cells from one healthy donor were used as effectors, and K562 (suspension) and IMR32 (adherent) were used as the target cells. Both target cells were first stained with Calcein AM, and seeded at 10,000 cells/well in a standard 96-well microplate. The donor NK cells were then added to each well at Effector-to-Target (E:T) ratios 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125:1. The 96 well plate was then scanned and analyzed using Celigo imaging cytometer at t = 1, 2, 3, and 4 h to measure the% lysis of target cells. The results showed increasing% lysis as incubation time and E:T ratio increased. The propose Celigo imaging cytometry is an accurate and simple method for direct quantification of cytotoxicity. Citation Format: Leo Li-Ying Chan, Kelsey Rosbach, Srinivas S. Somanchi, Dean Lee. Quantification of natural killer cell-mediated cytotoxicity using Celigo imaging cytometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1303. doi:10.1158/1538-7445.AM2015-1303

High-throughput direct cell counting-based natural killer cell-mediated cytotoxicity assay using Celigo Imaging Cytometry (TUM2P.1027)

Abstract Cytotoxicity assays play a central role in studying the function of immune effector cells such as cytolytic T lymphocytes (CTL) and natural killer (NK) cells. Traditionally, cytotoxicity assays have been performed using 51Chromium (51Cr) and Calcein release assays. The assays involve labeling tumor cells (target) with radioisotope or fluorescent dyes, when the target cells are subjected to cytolysis by CTLs or NK cells, they releases the entrapped labels into the media upon lysis. These traditional methods may generate inconsistent results due to low sensitivity caused by poor loading efficiency and high spontaneous release of the reagents. In this work, we demonstrate a novel high-throughput direct cell counting method for cytotoxicity assay using the Celigo imaging cytometry method. Human NK cells from one healthy donor were used as effectors, and K562 and IMR32 were used as the target cells. Both target cells were first stained with Calcein AM, and seeded at in a 96-well microplate. NK cells were then added to each well at various Effector-to-Target (E:T) ratios. The 96 well plate was then scanned and analyzed using the Celigo to measure the % lysis of target cells at different time points. The results showed increasing % lysis as incubation time and E:T ratio increased. The propose Celigo imaging cytometry is an accurate and simple method for direct quantification of cytotoxicity, which can be an attractive method for both academic and clinical research.

Image cytometry method for measuring concentration and viability of primary cells used in cellular therapy (TECH1P.846)

Abstract Cellular therapy has become a major clinical research field that creates tailor-made medical treatments for many human diseases. Primary cells obtained from patients and mouse models often contain nonspecific particles such as red blood cells (RBC), platelets, and cellular debris, which can make the cell sample analysis difficult. To remove nonspecific particles that can interfere with analysis, a ficoll gradient separation or RBC lysis is routinely performed. Measurements of concentration and viability of the cell sample are necessary for clinical researchers to qualify the collected patient samples for research and downstream processing. In this work, we validated a fluorescence-based image cytometry method using acridine orange (AO) and propidium iodide (PI) to rapidly measure concentration and viability without the need for tedious purification protocols. Using the Cellometer Vision instrument we performed the viability and concentration analysis and validated our results using the traditional hemacytometer method for samples including but not limited to peripheral blood mononuclear cells, mononuclear cells, Leuko Pac, bone marrow, cord blood, whole blood, bronchoaveolar lavage, and primary murine samples. This image-based cytometer method can increase clinical research efficiency by eliminating the need for purification steps and for manual counting. Furthermore, it can eliminate the user-to-user variation, thus improving the accuracy of the cell analysis.

Automated enumeration and viability measurement of canine stromal vascular fraction cells using fluorescence-based image cytometry method.

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. 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, 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.

Mycophenolic acid affects basic functions of human keratinocytes in the IMPDH-dependent manner

Clinical studies suggest that the immunosuppressant MPA is associated with impaired wound healing. It is believed that the main cause of impairment is the inhibition of inflammatory response. However, it is unknown whether MPA may directly affect epidermal cells. The aim of our study was to examine the direct influence of mycophenolic acid, the selective blocker of de novo purine synthesis, on human epidermal keratinocyte morphology, proliferation, motile activity, and differentiation in in vitro culture. The number of keratinocytes cultured in the presence of MPA was counted and cell motility was measured by a time-lapse computer-aided method. Cell morphology was determined by flow and image cytometry methods. Real-time RT-PCR analysis was employed to investigate the expression of markers of differentiation. We showed that MPA induces irreversible inhibition of cell proliferation, causes cell enlargement and impairs cell locomotion in a time-dependent manner. The level of expression of differentiation markers was significantly reduced by MPA treatment. All these effects were reversed by the addition of guanine. Our results indicated that MPA impairs basic functions of human skin keratinocytes via intracellular guanosine nucleotide depletion, which may be directly reflected in wound healing problems in patients treated with this immunosuppressant.

Automated quantification of budding Saccharomyces cerevisiae using a novel image cytometry method

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.

Phenolphthalein induces centrosome amplification and tubulin depolymerization in vitro

Aneuploidy is a major cause of human reproductive failure and plays a large role in cancer. Phenolphthalein (PHT) induces tumors in rodents but its primary mechanism does not seem to be DNA damage. In heterozygous TSG-p53(®) mice, PHT induces lymphomas and also micronuclei (MN), many containing kinetochores (K), implying chromosome loss (aneuploidy). The induction of aneuploidy would be compatible with the loss of the normal p53 gene seen in the lymphomas. In this study, we confirm PHT's aneugenicity and determine the aneugenic mechanism of PHT by combining traditional genetic toxicology assays with image and flow cytometry methods. The data revealed that PHT induces tubulin polymerization abnormalities and deregulates the centrosome duplication cycle causing centrosome amplification. We also show that one of the consequences of these events is apoptosis.

Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error (P3233)

Abstract 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. 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). 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. 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.

Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error

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.