Issue Of Cytometry Research Articles

Issue Highlights — May 2021

Issue Highlights — May 2021

In Memoriam: Mortimer L. Mendelsohn: (1 December, 1925-8 January, 2020).

In Memoriam: Mortimer L. Mendelsohn: (1 December, 1925-8 January, 2020).

ISSUE HIGHLIGHTS - July 2020.

ISSUE HIGHLIGHTS - July 2020.

Issue Highlights - November 2018.

Issue Highlights - November 2018.

γH2AX is a biomarker of modulated cytostatic drug resistance.

ONE reason for reduced therapeutic efficiency of cancer chemotherapies is the appearance of multidrug resistance. Plasma membrane glycoprotein (P-gp) is recognized as one of the best characterized drug efflux pumps playing a central role in the absorption and disposition of many drugs, including chemotherapeutic agents. Increased P-gp activity has been suggested and reported to be an indicator of chemotherapeutic failure. Therefore, P-gp activity has been used as a potential target in being able to reverse multidrug resistance. Cytostatic drugs such as etoposide in combination with the immunosuppressive cyclosporine A may inhibit P-gp activity, ultimately resulting in intracellular enrichment of the targeted drug (1). One issue in cancer therapy involves the highly varying drug response among patients. To assess the multidrug resistance status for personalized cancer therapy, an assay that can provide results to monitor the cytostatic effect of antineoplastic agents in combination with different drugs is highly desirable. Many of these drugs, like etoposide, cause DNA double strand breaks (DSBs) that may eventuate in clonogenic death. DSBs are one of the most biologically significant DNA damage lesions that leads to chromosome breakage and/or rearrangement, mutagenesis and loss or gain of genetic information (2). The phosphorylation of H2AX histone proteins which are located in the vicinity of the DSBs is known as one of the earliest responses to DNA DSBs in cells. Induction of DNA DSBs in live mammalian cells triggers the phosphorylation of Ser139 in the SQ motif near the C-terminal of H2AX protein, which results in the phosphorylated form of H2AX, termed cH2AX. cH2AX signals appear as discrete nuclear “foci” which can be visualized and quantified by a number of methods including fluorescence microscopy and flow cytometry, following immunostaining procedures with fluorescence-coupled antibodies. The cH2AX foci counting approach has been used in numerous studies to assess the relationship between cH2AX foci removal and the rate of DSBs repair (3). Each DSB is represented by an individual cH2AX focus with a ratio of 1:1 and after successful repair of DSBs, the level of cH2AX foci decline over time in the presence of normal physiological repair mechanisms. The presence of persistent cH2AX indicates impaired DNA repair. Therefore, cH2AX quantification may prove to be a sensitive biomarker of DNA DSBs in human cells and has been suggested for biomonitoring tumor progression and treatment response (4). DSBs are directly generated by exogenous agents such as ionizing radiation and antitumor drugs (bleomycin, mitoxantrone, etoposide). Because mammalian cells respond to DSBs by activating a multitude of proteins involved in signalling and DNA repair pathways, the very nature of DSBs poses such a threat to cell survival that DNA damage checkpoint proteins may be activated to initiate cellular division arrest. This provides time for DNA repair to proceed before mitosis is completed or in the case of overwhelming damage, apoptosis ensues (5). Therefore, detection of accumulated cH2AX foci may be an efficient method for assessing multidrug resistance in cancer therapy as presented by Reddig et al. in this issue of Cytometry A (page XXX).

Affinity-bead-mediated acoustophoresis: a novel tool in cytometry.

ACOUSTOPHORESIS combined with biofunctionalized capture beads—a method called affinity-bead-mediated acoustophoresis—can be used for highly selective isolation of CD41 lymphocytes from peripheral blood progenitor cell products (PBPCS). This opens possibilities for improved hematopoietic stem cell transplantation. Acoustophoresis is a method based on the migration of suspended particles or cells in an acoustic field caused by the acoustic radiation force. When implemented in a flowthrough setting, acoustophoresis can be used for separation of particles from a fluid medium, or separation of particles having different size and acoustic properties (1). Over the last 10–15 years, acoustophoresis applied to microfluidic devices has been rapidly developing (2), primarily due to its simplicity and low cost relative alternative methods. For cytometry purposes, acoustophoresis-based technology was recently (in year 2010) implemented commercially for improving cell alignment, as an alternative to the standard sheath-flow alignment [Attune

Epigenetics by flow!

RECENT advances in the field of cancer epigenetics (heritable changes in gene expression that occur without alteration in DNA sequence) with the increased understanding of the mechanisms of epigenetic silencing of tumor suppressor genes and activation of oncogenes—island methylation patterns, histone modifications, and dysregulation of DNA binding proteins-present great promise for future identification of novel biomarkers of application to cancer detection, prognosis, and therapy response (1,2). In particular, histones are essential for chromatin stability. They undergo many modifications, which seem to play important roles in transcriptional regulation and therefore potentially oncogenic if, for example, deregulation leads to the loss of expression of a tumor suppressor gene. Indeed, anomalous patterns of histone modifications are recognized as a hallmark of cancer (3). Histone H3 phosphorylation at serine 10 (and serine 28) occurs at mitosis and the Aurora Kinases that perform this H3 phosphorylation are frequently implicated in cancer (4). Similarly, signatures of histone modifications patterns, such as trimethyl-k9H3, are associated with patient prognosis in acute myeloid leukemia (AML) (5). Epigenetic biomarkers can be detected in tissue samples as well as in biofluids. Chromatin immunoprecipitation (ChIP) using antibodies raised against individual histone marks represents a useful tool for identification of epigenetic biomarkers in basic research and highly specific ChIP grade antibodies are readily available (6). However, this technology does not allow the characterization of epigenetic patterns in heterogeneous clinical samples. Multiparameter flow cytometry, in contrast, allows one to evaluate the expression of markers of interest on a per cell bases. As early as the late 1990s, we developed flow cytometric techniques to identify modifications in cells undergoing mitosis (H3S10 phosphorylation) that have been proven useful not only to identify mitotic cells, but also to study molecular mechanisms associated with the G2 to M transition and as a tool to screen in vivo Aurora Kinase inhibitors (7,8). However, and as noted in the work by Watson et al. (page 78) in the present issue of Cytometry A, condensed chromatin may negatively affect accessibility of antibodies to histone modifications involved in epigenetic regulation. Initial attempts to detect histone marks by flow cytometry were done in cell lines by Obier et al. (9). The authors in the present report extended this work by utilizing antibodies available for ChIP and adapting a protocol that allowed simultaneous measurement of surface immune phenotype, light scatter, and intracellular antigens for normal and leukemic blood evaluation (10). Increased trimethylation of histone H3 Lysine 27 (H3K27me3) has also been associated with tumorigenesis since can silence tumor suppressor genes despite the drop in methylation of the gene’s CpG island (an event that normally activates genes) (11). Interestingly, this is one of the clinically relevant histone marks tested in the Watson’s paper. As presented, the overall MFIs of the leukemic blast samples compared to any of the normal blood cell population seems to indicate increased trimethylation in at least some of the AML samples despite the clear heterogeneity observed. Although, further refinements in the fixation/staining protocol may be needed, this study represents a genuinely novel approach to cancer epigenetics using flow cytometry as the analytical tool. One can only expect an increased number of additional antibodies and applications in the future. The innovative work by Watson et al. in this issue of Cytometry A describes an approach to epigenetics that allows for the study of heterogeneous cell populations on a cell-bycell bases. The authors took advantage of antibodies

Issue Highlights—January 2013

Issue Highlights—January 2013

Modernizing the MTT assay with microfluidic technology and image cytometry

THE combination of microfluidic technology and imaging provides a new set of cytometric tools that can be quantitative and highly informative. Microfluidic devices can be used to capture small numbers of cells and expose those cells to a unique set of conditions (e.g., growth factors, toxins, and shear forces) by controlling the chemical and physical environment around the cells. The processes used to capture cells and manipulate the environment can all be controlled ‘‘on-chip,’’ reducing the number of manual handling steps, allowing for automation, minimizing the quantities of reagents required. Because the microfluidic device can be frequently placed on a microscope stage, quantitative, time lapse imaging can be used to record the dynamic responses of the cells. In the extreme, large numbers of individual cells can be specifically manipulated all while images are acquired on an automated microscope. In this issue of Cytometry, Lim et al. (page 691) adapt the standard methyl tetrazolium (MTT) assay (1) to a microfluidic device and use automated imaging and image analysis to obtain single cell measurements. The MTT assay is a colorimetric assay that is typically performed in 96-well microtiter plates and at the end of the assay an absorbance measurement is made from each well using a plate reader. In routine applications, the MTT assay is used to assess cell viability or to measure cell proliferation. Because pharmaceutical agents or toxins can stimulate or inhibit cell growth, the MTT assay is frequently used as cytotoxicity assay. Typically, the treated and control cells are incubated for 2–4 h with the MTT reagent, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, at concentrations in the range of 1 mM. Metabolically active cells reduce the MTT reagent into purple formazan crystals, presumably through mitochondrial enzymes such as succinate dehydrogenase. Before reading the plate, the crystals are dissolved with sodium dodecyl sulfate, and then the absorbance is measured for each well. The results of the standard MTT assay are ambiguous because the absorbance measurement alone cannot be used to distinguish between changes in average enzyme activity of the cell population from changes in the number of cells that can occur by either cell proliferation or death. Furthermore, the

A brilliant new addition to the fluorescent probe toolbox

A brilliant new addition to the fluorescent probe toolbox

Publication of optimized multicolor immunofluorescence panels

Publication of optimized multicolor immunofluorescence panels

Measuring Antigen‐Specific Immune Responses, 2008 update

Overall, the last 10 years have seen an explosion in the field of antigen-specific immune response monitoring. As summarized in this issue of Cytometry and at the MASIR conferences, these technologies have provided new insights into the basic biology of the immune system and are beginning to provide useful clinical information. These papers highlight the areas that are being actively explored.

Raman spectroscopy comes to flow cytometry

Raman spectroscopy comes to flow cytometry

All that glitters is not gold: All that FLICA binds is not caspase. A caution in data interpretation—and new opportunities

variety of cytometric methods that found application in cellnecrobiology have been developed during the past two decades(review, 1). One such methodology is based on use of thefluorochrome-labeled inhibitors of caspases (FLICA; alsoknown under names CaspaTag, CaspACE, CaspGLOW, orFLIVO). FLICA were designed as affinity ligands to enzymeactive centers to detect activation of caspases (2–4). Throughthe reactive fluoromethylketone (fmk) moiety they covalentlybind to cysteine of the active center of caspase to form a thio-methyl ketone and thereby irreversibly inactivate the targetenzyme (4,5). The specificity towards individual caspases isprovided by the sequence of amino acids in the tetra-peptidemoiety (5). Carboxyfluorescein (FAM) (1–4), fluorescein(FITC) (6), or sulforhodamine serves as a florescent tag toreport its binding and localization in the cell. FLICA ligandshave proven to be convenient and reliable reporters of caspaseactivation and induction of apoptosis (1–4,6).Using FLICA reagents we observed that their binding tocells undergoing apoptosis was not fully consistent with theexpected specificity, namely, it was not restricted to enzymaticcenters of respective activated caspases (7). We postulated,therefore, that FLICA binding sites may involve other cell con-a minor fraction of total FLICA binding to apoptotic could beattributed to its interaction with the respective caspases (7).The elegant studies of Kuzelova et al. in this issue of Cyto-metry (8) provide further evidence of the contribution of cellconstituents other than caspases in FLICA binding. Theauthors observed that extent of staining of apoptotic cells withFAM-DEVD-fmk was minimally changed after pretreatmentwith the unlabeled z-DEVD-fmk under conditions when cas-pase-3 activity was inhibited. Yet, FAM-DEVD-fmk boundirreversibly to large subunit of caspase-3 and its binding wasprevented by z-DEVD-fmk, as detected by Western blotting.FLICA, thus, binds to the enzymatic active center of their re-spective activated caspases, but this contribution is minimalwith respect to the overall cell staining. While the extent ofFLICA binding by cell constituents other than caspases mayvary between cell types and may also depend on inducer ofapoptosis, the data of Kuzelova et al. (8) and our findings (7)reveal that specificity of staining of individual caspases withFLICA is far from that of the originally anticipated or adver-tised by the vendors. These findings (7,8) call for re-examina-tion of interpretation of the data obtained with these reagents.While FLICA binding reports caspase activation and is amarker of apoptosis, the overall fluorescence intensity of apo-ptotic cell labeled with this ligand does not represent its bind-ing to enzyme active center of caspases. The same cautionshould be exercised interpreting data obtained with the use of

Preface

Preface