Systematic review and meta-analysis of cryopreserved bovine sperm assessment: harnessing imaging flow cytometry for multi-parametric analysis.

Cryopreservation of sperm is an essential technique in assisted reproduction in cattle. The objective of the study was to systematically review and synthesize the literature on bull semen quality evaluation based on the comparison of morphological and metabolic parameters of cryopreserved bovine spermatozoa such as DNA integrity, mitochondrial status, plasma membrane alterations, total motility, and morphology (% of abnormal cells). The electronic databases PubMed, Web of Sciences, Scopus, and Google Scholar were searched up to December 2023. Studies and references were included if they reported the following parameters: DNA integrity, mitochondrial status, plasma membrane alterations, total motility, and morphological aberrations (% of abnormal cells) for conventional cryopreserved bovine spermatozoa. After an electronic search, out of 1,526 original studies, only 40 were included in the meta-analysis. Standardized mean differences (SMD) with 95% confidence intervals were estimated for the chosen studies, and a meta-analysis was performed using a random effects model. The tau-squared (tau2) and inconsistency index (I2) quantified heterogeneity among different studies. The regression analysis for the evaluated parameters showed a positive correlation between mitochondrial membrane potential (MMP), total motility, and abnormal morphology and a negative correlation between DNA fragmentation index (DFI) and total motility and MMP. Moreover, subgroup analysis demonstrated similar associations for dairy and non-dairy bull breeds, albeit with lower I2 values. The presence of publication bias was confirmed by Egger's test, except for the MMP parameter. A multi-parametric analysis of morphological and metabolic parameters can address the existing limitations of cryopreserved bovine spermatozoa quality assessment. Combining imaging flow cytometry (IFC) with standardization of sperm pre-processing and optimization of the experimental protocols may help to differentiate sperm from cellular debris and cytoplasmic droplets of similar size and alleviate limitations demonstrated by conventional sperm analysis.

Using Virtual Filtering Approach to Discriminate Microalgae by Spectral Flow Cytometer.

Fluorescence methods are widely used for the study of marine and freshwater phytoplankton communities. However, the identification of different microalgae populations by the analysis of autofluorescence signals remains a challenge. Addressing the issue, we developed a novel approach using the flexibility of spectral flow cytometry analysis (SFC) and generating a matrix of virtual filters (VF) which allowed thorough examination of autofluorescence spectra. Using this matrix, different spectral emission regions of algae species were analyzed, and five major algal taxa were discriminated. These results were further applied for tracing particular microalgae taxa in the complex mixtures of laboratory and environmental algal populations. An integrated analysis of single algal events combined with unique spectral emission fingerprints and light scattering parameters of microalgae can be used to differentiate major microalgal taxa. We propose a protocol for the quantitative assessment of heterogenous phytoplankton communities at the single-cell level and monitoring of phytoplankton bloom detection using a virtual filtering approach on a spectral flow cytometer (SFC-VF).

Development of Spectral Imaging Cytometry.

Spectral flow cytometry is a new technology that enables measurements of fluorescent spectra and light scattering properties in diverse cellular populations with high precision. Modern instruments allow simultaneous determination of up to 40+ fluorescent dyes with heavily overlapping emission spectra, discrimination of autofluorescent signals in the stained specimens, and detailed analysis of diverse autofluorescence of different cells-from mammalian to chlorophyll-containing cells like cyanobacteria. In this paper, we review the history, compare modern conventional and spectral flow cytometers, and discuss several applications of spectral flow cytometry.

Imaging Flow Cytometry of Multi-Nuclearity.

Multi-nuclearity is a common feature for cells in different cancers. Also, analysis of multi-nuclearity in cultured cells is widely used for evaluating the toxicity of different drugs. Multi-nuclear cells in cancer and under drug treatments form from aberrations in cell division and/or cytokinesis. These cells are a hallmark of cancer progression, and the abundance of multi-nucleated cells often correlates with poor prognosis.The use of standard bright field or fluorescent microscopy to analyze multi-nuclearity at the quantitative level is laborious and can suffer from user bias. Automated slide-scanning microscopy can eliminate scorer bias and improve data collection. However, this method has limitations, such as insufficient visibility of multiple nuclei in the cells attached to the substrate at low magnification.Since quantification of multi-nuclear cells using microscopic methods might be difficult, imaging flow cytometry (IFC) is a method of choice for this. We describe the experimental protocol for the preparation of the samples of multi-nucleated cells from the attached cultures and the algorithm for the analysis of these cells by IFC. Images of multi-nucleated cells obtained after mitotic arrest induced by taxol, as well as cells obtained after cytokinesis blockade by cytochalasin D treatment, can be acquired at a maximal resolution of IFC. We suggest two algorithms for the discrimination of single-nucleus and multi-nucleated cells. The advantages and disadvantages of IFC analysis of multi-nuclear cells in comparison with microscopy are discussed.

Optimizing FlowCam Imaging Flow Cytometry Operation for Classification and Quantification of Microcystis Morphospecies.

Microcystis is a globally known cyanobacterium causing potentially toxic blooms worldwide. Different morphospecies with specific morphological and physiological characters usually co-occur during blooming, and their quantification employing light microscopy can be time-consuming and problematic. A benchtop imaging flow cytometer (IFC) FlowCam (Yokogawa Fluid Imaging Technologies, USA) was used to identify and quantitate different Microcystis morphospecies from environmental samples. We describe here the FlowCam methodology for sample processing and analysis of five European morphospecies of Microcystis common to the temperate zone. The FlowCam technique allows detection of different Microcystis morphospecies providing objective qualitative and quantitative data for statistical analysis.

Freshwater Cyanobacterial Toxins, Cyanopeptides and Neurodegenerative Diseases.

Cyanobacteria produce a wide range of structurally diverse cyanotoxins and bioactive cyanopeptides in freshwater, marine, and terrestrial ecosystems. The health significance of these metabolites, which include genotoxic- and neurotoxic agents, is confirmed by continued associations between the occurrence of animal and human acute toxic events and, in the long term, by associations between cyanobacteria and neurodegenerative diseases. Major mechanisms related to the neurotoxicity of cyanobacteria compounds include (1) blocking of key proteins and channels; (2) inhibition of essential enzymes in mammalian cells such as protein phosphatases and phosphoprotein phosphatases as well as new molecular targets such as toll-like receptors 4 and 8. One of the widely discussed implicated mechanisms includes a misincorporation of cyanobacterial non-proteogenic amino acids. Recent research provides evidence that non-proteinogenic amino acid BMAA produced by cyanobacteria have multiple effects on translation process and bypasses the proof-reading ability of the aminoacyl-tRNA-synthetase. Aberrant proteins generated by non-canonical translation may be a factor in neuronal death and neurodegeneration. We hypothesize that the production of cyanopeptides and non-canonical amino acids is a more general mechanism, leading to mistranslation, affecting protein homeostasis, and targeting mitochondria in eukaryotic cells. It can be evolutionarily ancient and initially developed to control phytoplankton communities during algal blooms. Outcompeting gut symbiotic microorganisms may lead to dysbiosis, increased gut permeability, a shift in blood-brain-barrier functionality, and eventually, mitochondrial dysfunction in high-energy demanding neurons. A better understanding of the interaction between cyanopeptides metabolism and the nervous system will be crucial to target or to prevent neurodegenerative diseases.

Association of Lung Fibrotic Changes and Cardiological Dysfunction with Comorbidities in Long COVID-19 Cohort.

Background. Long COVID-19 symptoms appeared in many COVID-19 survivors. However, the prevalence and symptoms associated with long COVID-19 and its comorbidities have not been established. Methods. In total, 312 patients with long COVID-19 from 21 primary care centers were included in the study. At the six-month follow-up, their lung function was assessed by computerized tomography (CT) and spirometry, whereas cardiac function was assessed by elec-trocardiogram (ECG), Holter ECG, echocardiography, 24 h blood pressure monitoring, and a six-minute walk test (6MWT). Results. Of the 312 persons investigated, significantly higher sys-tolic and diastolic blood pressure, left ventricular hypertrophy, and elevated NT-proBNP were revealed in participants with hypertension or type 2 diabetes. Left ventricular diastolic dysfunc-tion was more frequently present in patients with hypertension. The most common registered CT abnormalities were fibrotic changes (83, 36.6%) and mediastinal lymphadenopathy (23, 10.1%). Among the tested biochemical parameters, three associations were found in long COVID-19 patients with hypertension but not diabetes: increased hemoglobin, fibrinogen, and ferritin. Nine patients had persisting IgM antibodies to SARS-CoV-2. Conclusions. We demon-strated a strong association between signs of cardiac dysfunction and lung fibrotic changes with comorbidities in a cohort of long COVID-19 subjects.

To Die or Not to Die-Regulated Cell Death and Survival in Cyanobacteria.

Regulated cell death (RCD) is central to the development, integrity, and functionality of multicellular organisms. In the last decade, evidence has accumulated that RCD is a universal phenomenon in all life domains. Cyanobacteria are of specific interest due to their importance in aquatic and terrestrial habitats and their role as primary producers in global nutrient cycling. Current knowledge on cyanobacterial RCD is based mainly on biochemical and morphological observations, often by methods directly transferred from vertebrate research and with limited understanding of the molecular genetic basis. However, the metabolism of different cyanobacteria groups relies on photosynthesis and nitrogen fixation, whereas mitochondria are the central executioner of cell death in vertebrates. Moreover, cyanobacteria chosen as biological models in RCD studies are mainly colonial or filamentous multicellular organisms. On the other hand, unicellular cyanobacteria have regulated programs of cellular survival (RCS) such as chlorosis and post-chlorosis resuscitation. The co-existence of different genetically regulated programs in cyanobacterial populations may have been a top engine in life diversification. Development of cyanobacteria-specific methods for identification and characterization of RCD and wider use of single-cell analysis combined with intelligent image-based cell sorting and metagenomics would shed more light on the underlying molecular mechanisms and help us to address the complex colonial interactions during these events. In this review, we focus on the functional implications of RCD in cyanobacterial communities.

Changes in Phytoplankton Community Composition and Phytoplankton Cell Size in Response to Nitrogen Availability Depend on Temperature.

The climate-driven changes in temperature, in combination with high inputs of nutrients through anthropogenic activities, significantly affect phytoplankton communities in shallow lakes. This study aimed to assess the effect of nutrients on the community composition, size distribution, and diversity of phytoplankton at three contrasting temperature regimes in phosphorus (P)-enriched mesocosms and with different nitrogen (N) availability imitating eutrophic environments. We applied imaging flow cytometry (IFC) to evaluate complex phytoplankton communities changes, particularly size of planktonic cells, biomass, and phytoplankton composition. We found that N enrichment led to the shift in the dominance from the bloom-forming cyanobacteria to the mixed-type blooming by cyanobacteria and green algae. Moreover, the N enrichment stimulated phytoplankton size increase in the high-temperature regime and led to phytoplankton size decrease in lower temperatures. A combination of high temperature and N enrichment resulted in the lowest phytoplankton diversity. Together these findings demonstrate that the net effect of N and P pollution on phytoplankton communities depends on the temperature conditions. These implications are important for forecasting future climate change impacts on the world's shallow lake ecosystems.

Meta-Analysis and Systematic Review of Coagulation Disbalances in COVID-19: 41 Studies and 17,601 Patients.

Introduction: Coagulation parameters are important determinants for COVID-19 infection. We conducted meta-analysis to assess the association between early hemostatic parameters and infection severity. Methods: Electronic search was made for papers that addressed clinical characteristics of COVID-19 patients and disease severity. Results were filtered using exclusion and inclusion criteria and then pooled into a meta-analysis to estimate the standardized mean difference (SMD) with 95% confidence interval (CI) for D-dimers, fibrinogen, prothrombin time, platelet count (PLT), activated partial thromboplastin time. To explore the heterogeneity and robustness of our fundings, sensitivity and subgroup analyses were conducted. Publication bias was assessed with contour-enhanced funnel plots and Egger's test by linear regression. Coagulation parameters data from retrospective cohort study of 451 patients with COVID-19 at National Research Center for Cardiac Surgery were included in meta-analysis of published studies. Results: Overall, 41 original studies (17,601 patients) on SARS-CoV-2 were included. For the two groups of patients, stratified by severity, we identified that D-dimers, fibrinogen, activated partial thromboplastin time, and prothrombin time were significantly higher in the severe group [SMD 0.6985 with 95%CI (0.5155; 0.8815); SMD 0.661 with 95%CI (0.3387; 0.9833); SMD 0.2683 with 95%CI (0.1357; 0.4009); SMD 0.284 with 95%CI (0.1472; 0.4208)]. In contrast, PLT was significantly lower in patients with more severe cases of COVID-19 [SMD -0.1684 with 95%CI (-0.2826; -0.0542)]. Neither the analysis by the leave-one-out method nor the influence diagnostic have identified studies that solely cause significant change in the effect size estimates. Subgroup analysis showed no significant difference between articles originated from different countries but revealed that severity assessment criteria might have influence over estimated effect sizes for platelets and D-dimers. Contour-enhanced funnel plots and the Egger's test for D-dimers and fibrinogen revealed significant asymmetry that might be a sign of publication bias. Conclusions: The hemostatic laboratory parameters, with exception of platelets, are significantly elevated in patients with severe COVID-19. The two variables with strongest association to disease severity were D-dimers and fibrinogen levels. Future research should aim outside conventional coagulation tests and include analysis of clotting formation and platelet/platelet progenitors characteristics.

Comparative analysis of freshwater phytoplankton communities in two lakes of Burabay National Park using morphological and molecular approaches.

We analyzed phytoplankton assemblages' variations in oligo-mesotrophic Shchuchie and Burabay lakes using traditional morphological and next-generation sequencing (NGS) approaches. The total phytoplankton biodiversity and abundance estimated by both microscopy and NGS were significantly higher in Lake Burabay than in Lake Shchuchie. NGS of 16S and 18S rRNA amplicons adequately identify phytoplankton taxa only on the genera level, while species composition obtained by microscopic examination was significantly larger. The limitations of NGS analysis could be related to insufficient coverage of freshwater lakes phytoplankton by existing databases, short algal sequences available from current instrumentation, and high homology of chloroplast genes in eukaryotic cells. However, utilization of NGS, together with microscopy allowed us to perform a complete taxonomic characterization of phytoplankton lake communities including picocyanobacteria, often overlooked by traditional microscopy. We demonstrate the high potential of an integrated morphological and molecular approach in understanding the processes of organization in aquatic ecosystem assemblages.

Semi-automated classification of colonial Microcystis by FlowCAM imaging flow cytometry in mesocosm experiment reveals high heterogeneity during seasonal bloom.

A machine learning approach was employed to detect and quantify Microcystis colonial morphospecies using FlowCAM-based imaging flow cytometry. The system was trained and tested using samples from a long-term mesocosm experiment (LMWE, Central Jutland, Denmark). The statistical validation of the classification approaches was performed using Hellinger distances, Bray-Curtis dissimilarity, and Kullback-Leibler divergence. The semi-automatic classification based on well-balanced training sets from Microcystis seasonal bloom provided a high level of intergeneric accuracy (96-100%) but relatively low intrageneric accuracy (67-78%). Our results provide a proof-of-concept of how machine learning approaches can be applied to analyze the colonial microalgae. This approach allowed to evaluate Microcystis seasonal bloom in individual mesocosms with high level of temporal and spatial resolution. The observation that some Microcystis morphotypes completely disappeared and re-appeared along the mesocosm experiment timeline supports the hypothesis of the main transition pathways of colonial Microcystis morphoforms. We demonstrated that significant changes in the training sets with colonial images required for accurate classification of Microcystis spp. from time points differed by only two weeks due to Microcystis high phenotypic heterogeneity during the bloom. We conclude that automatic methods not only allow a performance level of human taxonomist, and thus be a valuable time-saving tool in the routine-like identification of colonial phytoplankton taxa, but also can be applied to increase temporal and spatial resolution of the study.

Neuronal extracellular microRNAs miR-124 and miR-9 mediate cell-cell communication between neurons and microglia.

In contrast to peripheral macrophages, microglia in the central nervous system (CNS) exhibit a specific deactivated phenotype; however, it is not clear how this phenotype is maintained. Two alternative hypotheses were postulated recently: (a) microglia differ from peripheral macrophages being derived from the yolk sac (YS), whereas peripheral macrophages originate from bone marrow (BM); (b) microglia acquire a specific phenotype under the influence of the CNS microenvironment. We have previously shown that microglia express miR-124, which was also induced in BM-derived macrophages co-cultured with a neurons. We here investigated the possibility of horizontal transfer of the neuron-specific microRNAs miR-124 and miR-9 from primary neurons to microglia/macrophages. We found that after incubation with neuronal conditioned media (NCM), macrophages downregulated activation markers MHC class II and CD45. Neither cultured adult microglia nor YS- and BM-derived macrophages demonstrated intrinsic levels of miR-124 expression. However, after incubation with NCM, miR-124 was induced in both YS- and BM-derived macrophages. Biochemical analysis demonstrated that the NCM contained miR-124 and miR-9 in complex with small proteins, large high-density lipoproteins (HDLs), and exosomes. MiR-124 and miR-9 were promptly released from neurons, and this process was inhibited by tetrodotoxin, indicating an important role of neuronal electric activity in secretion of these microRNAs. Incubation of macrophages with exogenous miR-124 resulted in efficient translocation of miR-124 into the cytoplasm. This study demonstrates an important role of neuronal miRNAs in communication of neurons with microglia, which favors the hypothesis that microglia acquire a specific phenotype under the influence of the CNS microenvironment.

Myeloid Cells in Intact Human Cervical Explants Capture HIV and Can Transmit It to CD4 T Cells.

The importance of myeloid cells in HIV transmission in the female genital tract is uncertain. Because it is difficult to study the early events in HIV transmission in humans, most of our knowledge is based on animal models of SIV infection in Rhesus macaques and more recently HIV infection in humanized mice. However, these models may not accurately recapitulate transmission in the human genital tract. CD14+ myeloid cells are the most abundant hematopoietic cells in the human cervical mucosa, comprising 40-50% of CD45+ mononuclear cells. Most CD14+ cells are CD14+CD11c- macrophages and about a third are CD14+CD11c+ tissue dendritic cells, which express the HIV-binding receptors, DC-SIGN and CX3CR1. To examine the role of mucosal myeloid cells in HIV transmission, we infected intact healthy human cervical explants with CCR5-tropic HIV-1 ex vivo and then sorted populations of cervical immune cells 20 h later to determine whether they took up virus and could transmit it to activated CD4 T cells. Viral RNA was detected in CD14+ myeloid cells in all but one of 10 donor tissue samples, even when HIV RNA was not detected in CD4+ T cells. HIV RNA was detected predominantly in CD14+CD11c+ dendritic cells rather than in CD14+CD11c- macrophages. The reverse transcriptase inhibitor, nevirapine, reduced HIV RNA in CD4+ T cells, but not in CD14+ cells. Moreover, integrated HIV DNA were not detected above background in myeloid cells but was detected in T cells. These data suggest that although HIV replicates in T cells, myeloid cells in the female genital mucosa capture viral particles, but do not replicate the virus at early timepoints. However, sorted CD14+ myeloid cells isolated 20 h post-infection from 5 HIV-infected cervical explants tested all transmitted HIV to activated CD4+ T cells, while only 1 sample of sorted CD4+ T cells did. Thus, myeloid cells in human cervical tissue capture HIV and are an important early cellular storage site of infectious virus.

Platelets mediate protective neuroinflammation and promote neuronal plasticity at the site of neuronal injury.

It is generally accepted that inflammation within the CNS contributes to neurodegeneration after traumatic brain injury (TBI), but it is not clear how inflammation is initiated in the absence of infection and whether this neuroinflammation is predominantly beneficial or detrimental. We have previously found that brain-enriched glycosphingolipids within neuronal lipid rafts (NLR) induced platelet degranulation and secretion of neurotransmitters and pro-inflammatory factors. In the present study, we compared TBI-induced inflammation and neurodegeneration in wild-type vs. St3gal5 deficient (ST3-/-) mice that lack major CNS-specific glycosphingolipids. After TBI, microglial activation and CNS macrophage infiltration were substantially reduced in ST3-/- animals. However, ST3-/- mice had a larger area of CNS damage with marked neuronal/axonal loss. The interaction of platelets with NLR stimulated neurite growth, increased the number of PSD95-positive dendritic spines, and intensified neuronal activity. Adoptive transfer and blocking experiments provide further that platelet-derived serotonin and platelet activating factor plays a key role in the regulation of sterile neuroinflammation, hemorrhage and neuronal plasticity after TBI.

Cyclic AMP Pathway Suppress Autoimmune Neuroinflammation by Inhibiting Functions of Encephalitogenic CD4 T Cells and Enhancing M2 Macrophage Polarization at the Site of Inflammation.

Although it has been demonstrated that cAMP pathway affect both adaptive and innate cell functions, the role of this pathway in the regulation of T-cell-mediated central nervous system (CNS) autoimmune inflammation, such as in experimental autoimmune encephalomyelitis (EAE), remains unclear. It is also unclear how cAMP pathway affects the function of CD4 T cells in vivo at the site of inflammation. We found that adenylyl cyclase activator Forskolin besides inhibition of functions autoimmune CD4 T cells also upregulated microRNA (miR)-124 in the CNS during EAE, which is associated with M2 phenotype of microglia/macrophages. Our study further established that in addition to direct influence of cAMP pathway on CD4 T cells, stimulation of this pathway promoted macrophage polarization toward M2 leading to indirect inhibition of function of T cells in the CNS. We demonstrated that Forskolin together with IL-4 or with Forskolin together with IL-4 and IFNγ effectively stimulated M2 phenotype of macrophages indicating high potency of this pathway in reprogramming of macrophage polarization in Th2- and even in Th1/Th2-mixed inflammatory conditions such as EAE. Mechanistically, Forskolin and/or IL-4 activated ERK pathway in macrophages resulting in the upregulation of M2-associated molecules miR-124, arginase (Arg)1, and Mannose receptor C-type 1 (Mrc1), which was reversed by ERK inhibitors. Administration of Forskolin after the onset of EAE substantially upregulated M2 markers Arg1, Mrc1, Fizz1, and Ym1 and inhibited M1 markers nitric oxide synthetase 2 and CD86 in the CNS during EAE resulting in decrease in macrophage/microglia activation, lymphocyte and CD4 T cell infiltration, and the recovery from the disease. Forskolin inhibited proliferation and IFNγ production by CD4 T cells in the CNS but had rather weak direct effect on proliferation of autoimmune T cells in the periphery and in vitro, suggesting prevalence of indirect effect of Forskolin on differentiation and functions of autoimmune CD4 T cells in vivo. Thus, our data indicate that Forskolin has potency to skew balance toward M2 affecting ERK pathway in macrophages and indirectly inhibit pathogenic CD4 T cells in the CNS leading to the suppression of autoimmune inflammation. These data may have also implications for future therapeutic approaches to inhibit autoimmune Th1 cells at the site of tissue inflammation.

Heterogeneity of Metazoan Cells and Beyond: To Integrative Analysis of Cellular Populations at Single-Cell Level.

In this paper, we review some of the recent advances in cellular heterogeneity and single-cell analysis methods. In modern research of cellular heterogeneity, there are four major approaches: analysis of pooled samples, single-cell analysis, high-throughput single-cell analysis, and lately integrated analysis of cellular population at a single-cell level. Recently developed high-throughput single-cell genetic analysis methods such as RNA-Seq require purification step and destruction of an analyzed cell often are providing a snapshot of the investigated cell without spatiotemporal context. Correlative analysis of multiparameter morphological, functional, and molecular information is important for differentiation of more uniform groups in the spectrum of different cell types. Simplified distributions (histograms and 2D plots) can underrepresent biologically significant subpopulations. Future directions may include the development of nondestructive methods for dissecting molecular events in intact cells, simultaneous correlative cellular analysis of phenotypic and molecular features by hybrid technologies such as imaging flow cytometry, and further progress in supervised and non-supervised statistical analysis algorithms.

Spectral and Imaging Flow Cytometry in Phytoplankton Research.

Spectral and imaging flow cytometry are emerging technologies that allow quantifying spectral, fluorescent, and/or morphological parameters of heterogeneous cellular populations. The protocol describes a detailed step-by-step analysis of microalgae using these techniques and examples from our laboratory (Aphanizomenon sp., Cryptomonas pyrenoidifera, and Chlorella sp.). Moreover, the chapter will be helpful to scientists who want to perform spectral flow cytometry and apply principal component analysis.

Quantitation of IRF3 Nuclear Translocation in Heterogeneous Cellular Populations from Cervical Tissue Using Imaging Flow Cytometry.

Imaging flow cytometry (IFC) has become a powerful tool for studying the activation of transcriptional factors in heterogeneous cell populations in high-content imaging mode. With considerable interest to the clinical development of IFC, the question becomes how we can accelerate its application to solid tissues. We developed the first IFC-based procedure to quantify the nuclear translocation of interferon regulatory factor (IRF) 3, an important measure of induction of type I interferon antiviral response, in primary human immune cells including in solid tissues. After tissue digestion and protocol optimization by spectral flow cytometry, cell suspension is stained for intracellular IRF3 and acquired by IFC. Image analysis is performed using an optimized nuclear mask and similarity score parameter to correlate the location of IRF3 staining and a nuclear dye. The technique measures IRF3 activation at a single cell level and can detect small changes in the percent of activated cells providing objective quantitative data for statistical analysis.

Diagnostic Potential of Imaging Flow Cytometry.

Imaging flow cytometry (IFC) captures multichannel images of hundreds of thousands of single cells within minutes. IFC is seeing a paradigm shift from low- to high-information-content analysis, driven partly by deep learning algorithms. We predict a wealth of applications with potential translation into clinical practice.