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The use of anthracycline derivatives was approved for the treatment of a broad spectrum of human tumors (i.e., breast cancer). The need to test these drugs on cancer models has pushed the basic research to apply many types of in vitro assays, and, among them, the study of anthracycline-induced apoptosis was mainly based on the application of flow cytometry protocols. However, the chemical structure of anthracycline derivatives gives them a strong autofluorescence effect that must be considered when flow cytometry is used. Unfortunately, the guidelines on the analysis of anthracycline effects through flow cytometry are lacking. Therefore, in this study, we optimized the flow cytometry detection of doxorubicin and epirubicin-treated breast cancer cells. Their autofluorescence was assessed both by using conventional and imaging flow cytometry; we found that all the channels excited by the 488 nm laser were affected. Anthracycline-induced apoptosis was then measured via flow cytometry using the optimized setting. Consequently, we established a set of recommendations that enable the development of optimized flow cytometry settings when the in vitro assays of anthracycline effects are analyzed, with the final aim to reveal a new perspective on the use of those in vitro tests for the further implementation of precision medicine strategies in cancer.

Recently, the protective and/or pathological role of virus-specific T cells in SARS-CoV-2 infection has been the focus of many studies. We investigated the anti-spike IgG levels and SARS-CoV-2-specific T cells in 125 donors (90 vaccinated with four different vaccine platforms, 16 individuals with a previous natural infection, and 19 not vaccinated donors who did not report previous SARS-CoV-2 infections). Our data show that anti-spike IgG titers were similar between naturally infected subjects and those vaccinated with adenoviral vector vaccines. Of note, all immunized donors produced memory CD4+ and/or CD8+ T cells. A sustained polyfunctionality of SARS-CoV-2-specific T cells in all immunized donors was also demonstrated. Altogether, our data suggest that the natural infection produces an overall response like that induced by vaccination. Therefore, this detailed immunological evaluation may be relevant for other vaccine efforts especially for the monitoring of novel vaccines effective against emerging virus variants. [ABSTRACT FROM AUTHOR]

Recently, the protective and/or pathological role of virus-specific T cells in SARS-CoV-2 infection has been the focus of many studies. We investigated the anti-spike IgG levels and SARS-CoV-2-specific T cells in 125 donors (90 vaccinated with four different vaccine platforms, 16 individuals with a previous natural infection, and 19 not vaccinated donors who did not report previous SARS-CoV-2 infections). Our data show that anti-spike IgG titers were similar between naturally infected subjects and those vaccinated with adenoviral vector vaccines. Of note, all immunized donors produced memory CD4+ and/or CD8+ T cells. A sustained polyfunctionality of SARS-CoV-2-specific T cells in all immunized donors was also demonstrated. Altogether, our data suggest that the natural infection produces an overall response like that induced by vaccination. Therefore, this detailed immunological evaluation may be relevant for other vaccine efforts especially for the monitoring of novel vaccines effective against emerging virus variants.

The human immune system comprises a diverse array of cells involved in innate and adaptive immunity, and these immune cells coordinate immune responses against pathogens through intricate interactions. Multicolor flow cytometry is a powerful technique for qualitatively and quantitatively measuring the characteristics of immune cells, offering advantages, such as high-dimensional analysis, elucidation of cellular heterogeneity, understanding of pathogenesis, development of therapeutic strategies, and platform flexibility. Here, we demonstrate a new immunophenotyping panel that allows simultaneous evaluation of the characteristics of T and B cells. This panel enables tracking of changes in the immune status due to aging, environmental factors, pathogen infections, and vaccine administration. Additionally, it includes co-stimulatory molecules for assessing the activation state of immune cells and inhibitory checkpoint molecules for evaluating exhaustion status, thereby providing valuable insights into the features of human immune responses. These analyses contribute to understanding the pathophysiology of diseases and developing therapeutic strategies while offering crucial information for assessing the correlation of symptoms with infections and evaluating the efficacy of vaccines., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance.

Abstract: Multidimensional single‐cell analysis requires approaches to visualize complex data in intuitive 2D graphs. In this regard, t‐distributed stochastic neighboring embedding (tSNE) is the most popular algorithm for single‐cell RNA sequencing and cytometry by time‐of‐flight (CyTOF), but its application to polychromatic flow cytometry, including the recently developed 30‐parameter platform, is still under investigation. We identified differential distribution of background values between samples, generated by either background calculation or spreading error (SE), as a major source of variability in polychromatic flow cytometry data representation by tSNE, ultimately resulting in the identification of erroneous heterogeneity among cell populations. Biexponential transformation of raw data and limiting SE during panel development dramatically improved data visualization. These aspects must be taken into consideration when using computational approaches as discovery tools in large sets of samples from independent experiments or immunomonitoring in clinical trials. [ABSTRACT FROM AUTHOR]

Applications of available multidimensional flow cytometry software are compared to determine which are most appropriate to portray complex datasets using T cell polyfunctionality as a model. Advancement in flow cytometry reagents and instrumentation has allowed for simultaneous analysis of large numbers of lineage/functional immune cell markers. Highly complex datasets generated by polychromatic flow cytometry require proper analytical software to answer investigators' questions. A problem among many investigators and flow cytometry Shared Resource Laboratories (SRLs), including our own, is a lack of access to a flow cytometry‐knowledgeable bioinformatics team, making it difficult to learn and choose appropriate analysis tool(s). Here, we comparatively assess various multidimensional flow cytometry software packages for their ability to answer a specific biologic question and provide graphical representation output suitable for publication, as well as their ease of use and cost. We assessed polyfunctional potential of TCR‐transduced T cells, serving as a model evaluation, using multidimensional flow cytometry to analyze 6 intracellular cytokines and degranulation on a per‐cell basis. Analysis of 7 parameters resulted in 128 possible combinations of positivity/negativity, far too complex for basic flow cytometry software to analyze fully. Various software packages were used, analysis methods used in each described, and representative output displayed. Of the tools investigated, automated classification of cellular expression by nonlinear stochastic embedding (ACCENSE) and coupled analysis in Pestle/simplified presentation of incredibly complex evaluations (SPICE) provided the most user‐friendly manipulations and readable output, evaluating effects of altered antigen‐specific stimulation on T cell polyfunctionality. This detailed approach may serve as a model for other investigators/SRLs in selecting the most appropriate software to analyze complex flow cytometry datasets. Further development and awareness of available tools will help guide proper data analysis to answer difficult biologic questions arising from incredibly complex datasets. [ABSTRACT FROM AUTHOR]

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB ( n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples ( n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice. © 2015 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Advances in flow cytometry bioinformatics have resulted in a wide variety of clustering, classification and visualization techniques. To objectively evaluate the performance of such methods, common benchmarks such as the FlowCAP initiative have proven to be of great value. In this work, we report on a novel method, FloReMi, which was developed to tackle the most recent FlowCAP IV challenge. This challenge was formulated as a survival modeling problem, where participants were expected to design a model to predict the time until progression to AIDS for HIV patients. It is known that variability in progression rate cannot be fully predicted by simple CD4+ T cell counts. However, it is hypothesized that the immunopathogenesis established early in HIV already indicates the course of future disease. Adequately estimating the progression rate of HIV patients is crucial in their treatment. Using an automated pipeline to preprocess the data, and subsequently identify and select informative cell subsets, a survival regression method based on random survival forests was built, which obtained the best results of all submitted approaches to the FlowCAP IV challenge. © 2015 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

CD4 + T regulatory cells (Tregs) are a specialized subset of T lymphocytes, which promote immune homeostasis and tumor immunosuppression by restricting effector T cell immune responses. The characterization of context-specific Treg phenotypic heterogeneity is pivotal to determine their potential contributions to diseases. In the recent years, high-dimensional single-cell technologies, such as single-cell RNA sequencing, mass cytometry, or polychromatic flow cytometry, have played a central role in elucidating the heterogeneity of the Treg compartment at the cellular and molecular levels. Here we describe an example of high-dimensional flow cytometry analysis capable of defining an effector Treg subpopulation that positively correlates with cancer progression. Moreover, we provide a workflow template of high-dimensional single-cell analysis that is readily applicable to any leukocyte subpopulation., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The use of anthracycline derivatives was approved for the treatment of a broad spectrum of human tumors (i.e., breast cancer). The need to test these drugs on cancer models has pushed the basic research to apply many types of in vitro assays, and, among them, the study of anthracycline-induced apoptosis was mainly based on the application of flow cytometry protocols. However, the chemical structure of anthracycline derivatives gives them a strong autofluorescence effect that must be considered when flow cytometry is used. Unfortunately, the guidelines on the analysis of anthracycline effects through flow cytometry are lacking. Therefore, in this study, we optimized the flow cytometry detection of doxorubicin and epirubicin-treated breast cancer cells. Their autofluorescence was assessed both by using conventional and imaging flow cytometry; we found that all the channels excited by the 488 nm laser were affected. Anthracycline-induced apoptosis was then measured via flow cytometry using the optimized setting. Consequently, we established a set of recommendations that enable the development of optimized flow cytometry settings when the in vitro assays of anthracycline effects are analyzed, with the final aim to reveal a new perspective on the use of those in vitro tests for the further implementation of precision medicine strategies in cancer.

A subset of peripheral blood hematopoietic stem and progenitor cells of bone marrow origin is elevated in humans with solid cancers before treatment and declines with therapy. This biomarker of angiogenesis is not specific to tumor type and has great potential in the objective assessment of treatment response in clinical trials. This pilot study was designed to develop a biomarker of neoangiogenesis in dogs for the diagnosis of cancer, the measurement of treatment response, and the provision of objective data in clin-ical trials. Polychromatic flow cytometry was used to quantify two subsets of circulating hematopoietic stem and progenitor cells in dogs with spontaneous solid tumors before (n = 8) and after (n = 3) treat-ment, and normal controls (n = 6). Pro-angiogenic peripheral blood cells of bone marrow origin were detected in all eight cases and the six normal controls: however, there was no statistically significant difference between the two groups. Inter-estingly, an apparent decline in pro-angiogenic cells was observed after treatment. Bone marrow derived hematopoietic cells appear to contribute to tumor angiogenesis in dogs, as has been previously reported in humans. While the methodology for pro-angiogenic cell quantification in a small number of dogs in the current study did not result in a significant difference from normal controls, an optimized canine poly-chromatic flow cytometry protocol holds great promise in the development of a canine cancer model and for the objective measurements of treatment response in clinical trials. [ABSTRACT FROM AUTHOR]

Analysis of high-dimensional flow cytometry datasets can reveal novel cell populations with poorly understood biology. Following discovery, characterization of these populations in terms of the critical markers involved is an important step, as this can help to both better understand the biology of these populations and aid in designing simpler marker panels to identify them on simpler instruments and with fewer reagents (i.e., in resource poor or highly regulated clinical settings). However, current tools to design panels based on the biological characteristics of the target cell populations work exclusively based on technical parameters (e.g., instrument configurations, spectral overlap, and reagent availability). To address this shortcoming, we developed RchyOptimyx (cellular hieraRCHY OPTIMization), a computational tool that constructs cellular hierarchies by combining automated gating with dynamic programming and graph theory to provide the best gating strategies to identify a target population to a desired level of purity or correlation with a clinical outcome, using the simplest possible marker panels. RchyOptimyx can assess and graphically present the trade-offs between marker choice and population specificity in high-dimensional flow or mass cytometry datasets. We present three proof-of-concept use cases for RchyOptimyx that involve 1) designing a panel of surface markers for identification of rare populations that are primarily characterized using their intracellular signature; 2) simplifying the gating strategy for identification of a target cell population; 3) identification of a non-redundant marker set to identify a target cell population. © 2012 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Abstract: Flow cytometry has been the premier tool for single cell analysis since its invention in the 1960s. It has maintained this position through steady advances in technology and applications, becoming the main force behind interrogating the complexities of the immune system. Technology development was a three-pronged effort, including the hardware, reagents, and analysis algorithms to allow measurement of as many as 20 independent parameters on each cell, at tens of thousands of cells per second. In the coming years, cytometry technology will integrate with other techniques, such as transcriptomics, metabolomics, and so forth. Ongoing efforts are aimed at algorithms to analyse these aggregated datasaets over large numbers of samples. Here we review the development efforts heralding the next stage of flow cytometry. [Copyright &y& Elsevier]

Abstract: Danon disease (DD) is a monogenic X-linked disorder characterized by cardiomyopathy, skeletal myopathy and variable degrees of intellectual disability. DD develops due to mutations in the gene encoding lysosomal-associated membrane protein 2 (LAMP2). We report on a family exhibiting the clinical phenotype comprising of hypertrophic cardiomyopathy and ventricular pre-excitation, myopia and mild myopathy in two male patients and cardiomyopathy and myopia in a female patient. The diagnosis of DD in this family was based on the assessment of the clinical phenotypes and the absence of LAMP2 in skeletal and/or cardiac muscle biopsy specimens. Sequence analysis of the LAMP2 gene and its mRNA revealed a novel LAMP2 mutation (c.940delG) in all three patients. Approximately 25% of the female patient''s cardiomyocytes were LAMP2 positive apparently due to the unfavorable skewing of X chromosome inactivation. We further performed qualitative LAMP2 immunohistochemistry on peripheral white blood cells using the smear technique and revealed the absence of LAMP2 in the male patients. LAMP2 expression was further assessed in granulocytes, CD4+ and CD8+ T lymphocytes, CD20+ B lymphocytes, CD14+ monocytes and CD56+ natural killer cells by quantitative polychromatic flow cytometry. Whereas the male DD patients lacked LAMP2 in all WBC populations, the female patient expressed LAMP2 in 15.1% and 12.8% of monocytes and granulocytes, respectively. LAMP2 expression ratiometrics of highly vs. weakly expressing WBC populations discriminated the DD patients from the healthy controls. WBCs are thus suitable for initial LAMP2 expression testing when DD is a differential diagnostic option. Moreover, flow cytometry represents a quantitative method to assess the skewing of LAMP2 expression in female heterozygotes. Because LAMP2 is a major protein constituent of the membranes of a number of lysosome-related organelles, we also tested the exocytic capacity of the lytic granules from CD8+ T lymphocytes in the patient samples. The degranulation triggered by a specific stimulus (anti-CD3 antibody) was normal. Therefore, this process can be considered LAMP2 independent in human T cells. The c.940delG mutation results in a putatively truncated protein (p.A314QfsX32), which lacks the transmembrane domain and the cytosolic tail of the wild-type LAMP2. We tested whether this variant becomes exocytosed because of a failure in targeting to late endosomes/lysosomes. Western blotting of cardiac muscle, WBCs and cultured skin fibroblasts (and their culture media) showed no intra- or extracellular truncated LAMP2. By comparing the expression pattern and intracellular targeting in cultured skin fibroblasts of normal LAMP2 isoforms (A, B and C) tagged with green fluorescent protein (GFP) and the A314Qfs32-GFP fusion, we found that the A314Qfs32-GFP protein is not even expressed. These observations suggest that the truncated protein is unstable and is co-translationally or early post-translationally degraded. [Copyright &y& Elsevier]

The distribution of peripheral T cell subsets in young and healthy old people is markedly different, characterized by decreased numbers of naïve cells and increased numbers and clonal expansions of memory cells, predominantly in the CD8+ MHC class I-restricted subset. Here, however, we document dramatic alterations in naïve and memory subsets of CD4+ cells in patients with mild Alzheimer's disease (AD), with greatly decreased percentages of naïve cells, elevated memory cells, and increased proportions of CD4+ but not CD8+ cells lacking the important costimulatory receptor CD28. CD4+CD25^{high} potentially T regulatory cells with a naïve phenotype are also reduced in AD patients. Together these data provide stronger evidence than hitherto presented for more highly differentiated CD4+ as well as CD8+ T cells in AD patients, consistent with an adaptive immune system undergoing persistent antigenic challenge and possibly manifesting dysregulation as a result. [ABSTRACT FROM AUTHOR]

Abstract: The development of soluble recombinant peptide-major histocompatibility complex class I (pMHCI) molecules conjugated in multimeric form to fluorescent labels has enabled the physical quantification and characterization of antigen-specific CD8+ T cell populations by flow cytometry. Several factors determine the binding threshold that enables visualization of cognate CD8+ T cells with these reagents; these include the affinity of the T cell receptor (TCR) for pMHCI antigen. Here, we show that multimers constructed from peptide-human leukocyte antigen (pHLA) A⁎0201 monomers engineered in the heavy chain α2 domain to enhance CD8 binding (K D ≈85 μM) without impacting the TCR binding platform can detect cognate CD8+ T cells bearing low affinity TCRs that are not visible with the corresponding wildtype pHLA A⁎0201 multimeric complexes. Mechanistically, this effect is mediated by a disproportionate enhancement of the TCR/pMHCI association rate. In direct ex vivo applications, these coreceptor-enhanced multimers exhibit faithful cognate binding properties; concomitant increases in background staining within the non-cognate CD8+ T cell population can be resolved phenotypically using polychromatic flow cytometry as a mixture of naïve and memory cells. These findings provide the first validation of a novel approach to the physical detection of low avidity antigen-specific CD8+ T cell populations; such coreceptor-enhanced multimeric reagents are likely to be useful in a multitude of settings for the detection of auto-immune, tumor-specific and cross-reactive CD8+ T cells. [Copyright &y& Elsevier]

Abstract: The rapid progress in flow cytometry means that an increasing number of parameters can be looked at simultaneously, which is highly relevant for the assessment of lymphocyte characteristics, in particular their multifunctional profile. However, procedures using less complex technology need now optimization in order to take into account the new reagents and applications related to polychromatic flow cytometry. Through optimization of an immunomonitoring protocol to assess the functional profile of antigen specific T-cells using 9–10 colour flow cytometry, we tested the efficacy of three distinct standardized permeabilization buffers for the staining of relevant intracellular molecules. We show significant discrepancies in staining sensitivity for cytokine and cytotoxic factor expression from one permeabilization kit to another, which can lead to different data and interpretation. It is important to be aware of this potential bias and to design specific application/experimental procedures in order to obtain optimal results. [Copyright &y& Elsevier]

Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance.

Background: Multimer technology is widely used to screen antigen-specific immune recovery after allogeneic hematopoietic stem cell transplantation (allo-HSCT) as it enables identification, enumeration, phenotypic characterization and isolation of virus-specific T-cells. Novel approaches of multimerization might improve on classical tetramer staining; however, their use as standard monitoring technique to quantify antigen-specific cells has not been validated yet. We have compared two of these available multimeric complexes: pentamer and streptamer to select the best strategy for the incorporation into clinical monitoring practice., Methods: CMVpp65 495-503 -specific HLA-A*02:01 CD8 + T lymphocytes (CTL A * 02:01 -CMVpp65 495-503 ) were examined with pentamer and streptamer in peripheral blood cells of 77 healthy volunteers. Quantitative and qualitative analyses were performed to compare the precision and repeatability, sensitivity and accuracy and specificity of both technologies by flow cytometry., Results: Standard deviation for both techniques was less than 0.05 showing that they are repetitive and precise. Both techniques significantly correlated at high frequencies (r Spearman  = 0.9422; P < 0.0001) but it was lost at lower levels (<1%) of CTL A * 02:01 -CMVpp65 495-503 (r Spearman  = 0.3351; P = 0.1376). Streptamer is more accurate for the detection of CTL A * 02:01 -CMVpp65 495-503 providing significantly closer values to the theoretical ones (P < 0.0001) as pentamer binds unspecifically to a notable proportion of non-CMV-specific CD8 + T-cells., Conclusion: Our results suggest that streptamer multimer provides precise, accurate and specific results to detect CTL A * 02:01 -CMVpp65 495-503 by flow cytometry. Streptamer multimer can be used not only for the monitoring of early CTL A * 02:01 -CMVpp65 495-503 reconstitution in immunosuppressed patients following allo-HSCT but also, in conjunction with its reversibility role, for the isolation of CTL A * 02:01 -CMVpp65 495-503 for its future use in adoptive immunotherapy. © 2016 International Clinical Cytometry Society., (© 2016 International Clinical Cytometry Society.)

Flow cytometry is a powerful and robust technology for detecting and monitoring multiple markers at the level of single cells. Since its early development, flow cytometry has been used to assess heterogeneity in a cell suspension. Over the years, the increasing number of parameters that could be included in a single assay combined with physical separation by fluorescence-activated cell sorting (FACS) revealed that the T cell compartment is extremely heterogenous in terms of phenotypic diversity, functional capacity, and transcriptional regulation. While naïve T cells are fairly homogenous, diversity becomes extreme in the antigen-experienced memory compartment. The precise identification of memory subsets by the simultaneous analysis of multiple markers by flow cytometry is key not only to basic science but also for the correct immunomonitoring of patients undergoing immunotherapy or for T cell-based therapeutic approaches. In this chapter, we provide guidelines to optimize complex flow cytometry panels, to achieve correct fluorescence compensation and determine positivity for a given antigen. Correct selection of reagents and their validation is essential to the success of the assay. Despite having been developed for the identification of human naïve and memory T cell subsets, the concepts illustrated here can be applied to any experiment aiming to investigate n parameters by flow cytometry.

In signaling cascades downstream of NK cell activating receptor engagement, Ca(2+) ions are pivotal second messengers for NK cell cytotoxicity as well as cytokine production. Upon cellular activation, intracellular mobilization of Ca(2+) ions initially involves depletion of endoplasmic reticulum stores, leading to subsequent Ca(2+) influx through specific plasma membrane Ca(2+) release activated Ca(2+) channels. Multiple probes and assays for detecting intracellular Ca(2+) concentrations have been developed. With the advance of multiparameter flow cytometry instrumentation, a thorough analysis of signaling in specific NK cell subsets is possible. Here, a flow cytometric method for dynamic measurements of intracellular Ca(2+) concentrations in human NK cells subsets is detailed and discussed. This assay can be further adapted for specific scientific and diagnostic questions, with implications for various immunopathological conditions.

The lipophilic cation JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl carbocyanine iodide) has been used for more than 20 years as a specific dye for measuring mitochondrial membrane potential (ΔΨ(m)). In this unit, we revise our original protocol (that made use of a single 488 nm laser for the detection of monomers and aggregates, and where compensation was an important step) to use dual-laser excitation. Moreover, thanks to recently developed multilaser instruments and novel probes for surface and intracellular markers, JC-1 can be utilized by polychromatic flow cytometry to simultaneously detect, without any compensation between fluorescences, ΔΨ(m) along with other biological parameters, such as apoptosis and the production of reactive oxygen species., (Copyright © 2015 John Wiley & Sons, Inc.)

Flow cytometry and cell sorting are critical tools in stem cell research. Recent advances in flow cytometric hardware, reagents, and software have synergized to permit the stem cell biologist to more full identify and isolate rare cells based on their immunofluorescent and light scatter characteristics. Some of these improvements include physically smaller air-cooled lasers, new designs in optics, new fluorescent conjugate-excitation pairs, and improved software to visualize data, all which combine to open up new horizons in the study of stem cells, by enhancing the resolution and specificity of inquiry. In this review, these recent improvements in technology will be outlined and important cell surface and functional antigenic markers useful for the study of stem cells described.