Neutrophils are an important cellular component of the innate immune system that provides immediate protection to the host from infection. Neutrophil infiltration into inflamed peripheral tissues during infection is beneficial for immunity through phagocytosis of microbes, the release of antimicrobial factors, and secretion of proinflammatory cytokines. Recent reports further suggest that spleen-infiltrating neutrophils play a role in the adaptive immune response by providing survival signals to B cells. However, neutrophils may have detrimental effects on immunity in inflammatory diseases where their recruitment to lymphoid tissues and activation occur abnormally. To determine the contribution of neutrophils that reside in secondary lymphoid tissues to adaptive immunity, direct evaluation of the functional properties of tissue-resident neutrophils is required. We have developed a modified magnetic bead isolation approach for purifying neutrophils from inflamed spleens of autoimmune-prone mice by negative selection. Using this approach, we yielded neutrophils with greater than 90% purity without compromising cell viability. Equally important, the isolation procedure had little effect on the activation of neutrophils and did not impair phagocytic function. Thus, isolation of spleen-resident neutrophils by this optimized approach could be useful for interrogating the functional role of murine neutrophils in normal and abnormal immune responses.
magnetic bead isolation; murine neutrophil; spleen; inflammation; autoimmunity; flow cytometry
Alterations of blood rheology (hemorheology) are important for the early diagnosis, prognosis, and prevention of many diseases, including myocardial infarction, stroke, sickle cell anemia, thromboembolism, trauma, inflammation, and malignancy. However, real-time in vivo monitoring of hemorheological status using multiple parameters over long periods of time has not been reported. Here we describe the capability of label-free photoacoustic (PA) and photothermal (PT) flow cytometry in detection and imaging modes for dynamic monitoring of rheological parameters in circulating blood. We show that this integrated platform can simultaneously measure the main rheological parameters and may improve their diagnostic value. Using phenomenological approaches, we analyze correlations of PT and PA signal characteristics in the dynamic modes with red blood cell (RBC) aggregation, deformability, shape (e.g., as in sickle cells), intracellular hemoglobin distribution, individual cell velocity, flux of RBCs, and likely shear rate. Proof of concept is demonstrated in ex vivo and in vivo tests, including high-speed PT imaging of RBC shape in pathological conditions and identification of sickle cells in a mouse model of human sickle cell disease. These studies revealed the potential of this new platform integrating PT, PA, and conventional optical techniques for translation to use in humans using safe, portable, laser-based medical devices for point-of-care screening of disease progression and therapy efficiency.
Hematopoietic stem cells (HSCs) remain the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess functional potential. However, despite over 40 years of research, working with HSCs in the mouse remains challenging because of the relative abundance (or lack thereof) of these cells in the bone marrow. The frequency of HSCs in bone marrow is about 0.01% of total nucleated cells and approximately 5000 can be isolated from an individual mouse depending on the age, sex and strain of mice as well as purification scheme utilized. Adding to the challenge is the continual reporting of new markers for HSC purification, which makes it difficult for the uninitiated in the field to know which purification strategies yield the highest proportion of long-term, multi-lineage HSCs. Here, we will report will review different strategies for hematopoietic stem and progenitor purification identification. We will also discuss methods for rapid flow cytometric analysis of peripheral blood cell types, and novel strategies for working with rare cell populations such as HSCs in the analysis of cell cycle status by BrdU, Ki-67 and Pyronin Y staining. The purpose of this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology.
Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The present study describes approaches to delineate cell cycle stages utilizing iododeoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.
Mass Cytometry; Cell Cycle; Flow Cytometry; Retinoblastoma; iododeoxyuridine; hematopoiesis
We have developed a high throughput platform to detect the presence of HIV-1 and SIV-specific ADCC-mediating antibody responses. The assay is based on the hydrolysis of a cell-permeable fluorogenic peptide substrate containing a sequence recognized by the serine protease, Granzyme B (GzB). GzB is delivered into target cells by cytotoxic effector cells as a result of antigen (Ag)-specific Ab-Fcγ receptor interactions. Within the target cells, effector cell-derived GzB hydrolyzes the substrate, generating a fluorescent signal that allows individual target cells that have received a lethal hit to be identified by flow cytometry. Results are reported as the percentage of target cells with GzB activity (%GzB). Freshly isolated or cryopreserved PBMC and/or NK cells can be used as effector cells. CEM.NKR cells expressing the CCR5 co-receptor are used as a target cells following (i) coating with recombinant envelope glycoprotein, (ii) infection with infectious molecular clones expressing the Env antigens of primary and lab adapted viruses, or (iii) chronic infection with a variant of HIV-1/IIIB, termed A1953. In addition, primary CD4+ T cells infected with HIV-1 in vitro can also be used as targets. The assay is highly reproducible with a coefficient of variation of less than 25%. Target and effector cell populations, in the absence of serum/plasma, were used to calculate background (8.6±2.3%). We determined that an initial dilution of 1:50 and 1:100 is required for testing of human and non-human primate samples, respectively. This assay allows for rapid quantification of HIV-1 or SIV-specific ADCC-mediating antibodies that develop in response to vaccination, or in the natural course of infection, thus providing researchers with a new methodology for investigating the role of ADCC-mediating antibodies as correlates of control or prevention of HIV-1 and SIV infection.
ADCC; HIV; SIV; NK; Fc gamma receptors; Granzyme B; high throughput
We provide an overview of the methods used to label circulating cells for fluorescence detection by in vivo flow cytometry. These methods are useful for cell tracking in small animals without the need to draw blood samples, and are particularly useful for the detection of circulating cancer cells and quantification of circulating immune cells.
In vivo flow cytometry; cell tracking; circulating tumor cells; circulating immune cells
The Flow Cytometry Standard (FCS) format was developed back in 1984. Since then, FCS became the standard file format supported by all flow cytometry software and hardware vendors. Over the years, updates were incorporated to adapt to technological advancements in both flow cytometry and computing technologies. However, flexibility in how data may be stored in FCS has led to implementation difficulties for instrument vendors and third party software developers. In this technical note, we are providing implementation guidance and examples related to FCS 3.1, the latest version of the standard. By publishing this text, we intend to prevent potential compatibility issues that could be faced when implementing the FCS spillover and preferred display keywords that have arisen during discussions among some implementers.
flow cytometry; FCS; data standard; file format; bioinformatics
Purpose and Appropriate Sample Types
This panel was optimized to assess CD4+ and CD8+ T cell responses to various tumor antigens from melanoma patients. The panel was tested on single-cell derived T cell isolates (SCD-T) and T cell lines derived from peripheral blood mononuclear cells (PBMC) from melanoma patients, T cell lines from the tumor environment of melanoma patients, and fresh and cryopreserved PBMC (healthy donors). Staining can be performed in 96-well plates for high-throughput.
Immunophenotype; T cells; intracellular cytokine staining; melanoma
Flow cytometry has been a fundamental tool of biological discovery for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents studying cells in their native environment. These problems can be overcome by use of in vivo flow cytometry which provides detection and imaging of circulating normal and abnormal cells directlyin blood or lymph flow. The goal of this mini-review is to provide a brief history, features and challenges of this new generation of flow cytometry methods and instruments. Spectrum of possibilities of in vivo flow cytometry in biological science (e.g., cell metabolism, immune function, or apoptosis) and medical fields (e.g., cancer, infection, cardiovascular disorder) including integrated photoacoustic-photothermal theranostics of circulating abnormal cells are discussed with focus on recent advances of this new platform.
flow cytometry; spectral imaging; photoacoustic and photothermal methods; fluorescence; Raman spectroscopy; blood and lymph flows; circulating tumor cells; bacteria, and multicolor nanoparticles; theranostics
The androgen receptor (AR) is a steroid hormone receptor which regulates transcription of androgen-sensitive genes and is responsible for the development and maintenance of male secondary sexual characteristics. Chemicals that interfere with AR activity may lead to pathological conditions in androgen-sensitive tissues. A variety of reporter systems have been developed, driven by androgen sensitive promoters, which screen for chemicals that modulate androgenic activity. We have developed a flexible, high-throughput AR transcriptional activation assay, designated the Multifunctional Androgen Receptor Screening (MARS) assay, to facilitate the identification of novel modulators of AR transcriptional activity using flow cytometry.
Androgen-independent human prostate cancer-derived PC3 cells were transiently co-transfected with an expression vector for the wild-type human AR and an androgen-sensitive promoter regulating the expression of destabilized enhanced GFP (dsEGFP). The transfected cells were stimulated with established androgenic and antiandrogenic compounds and assessed for increased or decreased dsEGFP expression. To screen for antagonists of AR transcription, the AR agonist R1881 was co-administered at sub-maximal concentrations with potential AR antagonists. The assay was formatted for high throughput screening using the HyperCyt® flow cytometry system.
Agents with established androgenic and antiandrogenic activity were used for validation of the MARS assay. AR agonists were found to potently induce dsEGFP. Furthermore, AR agonists induced dsEGFP expression in a dose-dependent manner. Alternatively, AR antagonists blocked dsEGFP expression when co-administered with low-dose R1881, which also occurred in a dose-dependent manner.
Modulators of AR transcriptional activity can be successfully identified by the MARS assay, utilizing a rapid, flexible, sensitive, and high-throughput format. Dose-response curves can be successfully generated for these compounds, allowing for an assessment of potency. Due to its simplicity and high-throughput compatibility, the MARS assay and HyperCyt® system combined with flow cytometric analysis represents a valuable and novel addition to the current repertoire of AR transcriptional activation screening assays.
Androgen receptor assay; androgens; antiandrogens; flow cytometry; HyperCyt®; biomolecular screening
To study the process of morphogenesis, one often needs to collect and segment time-lapse images of living tissues to accurately track changing cellular morphology. This task typically involves segmenting and tracking tens to hundreds of individual cells over hundreds of image frames – a scale that would certainly benefit from automated routines; however, any automated routine would need to reliably handle a large number of sporadic, and yet typical problems (e.g., illumination inconsistency, photobleaching, rapid cell motions, drift of focus or of cells moving through the imaging plane). Here, we present a segmentation and cell tracking approach based on the premise that users know their data best – interpreting and using image features that are not accounted for in any a priori algorithm design. We have developed a program, SeedWater Segmenter (SWS), that combines a parameter-less and fast automated watershed algorithm with a suite of manual intervention tools that enables users with little to no specialized knowledge of image processing to efficiently segment images with near-perfect accuracy based on simple user interactions.
image segmentation; computer-assisted image processing; tissue analysis; confocal microscopy; Drosophila; embryogenesis
We present results from machine classification of melanoma biopsies sectioned and stained with hematoxylin/eosin (H&E) on tissue micro-arrays (TMA). The four stages of melanoma progression were represented by seven tissue types, including benign nevus, primary tumors with radial and vertical growth patterns (stage I) and four secondary metastatic tumors: subcutaneous (stage II), lymph node (stage III), gastrointestinal and soft tissue (stage IV). Our experiment setup comprised 14,208 image samples based on 164 TMA cores. In our experiments we constructed an HE color space by digitally deconvolving the RGB images into separate H (hematoxylin) and E (eosin) channels. We also compared three different classifiers: Weighted Neighbor Distance (WND), Radial Basis Functions (RBF), and k-Nearest Neighbors (kNN). We found that the HE color space consistently outperformed other color spaces with all three classifiers, while the different classifiers did not have as large of an effect on accuracy. This showed that a more physiologically relevant representation of color can have a larger effect on correct image interpretation than downstream processing steps. We were able to correctly classify individual fields of view with an average of 96% accuracy when randomly splitting the dataset into training and test fields. We also obtained a classification accuracy of 100% when testing entire cores that were not previously used in training (four random trials with one test core for each of 7 classes, 28 tests total). Because each core corresponded to a different patient, this test more closely mimics a clinically relevant setting where new patients are evaluated based on training with previous cases. The analysis method used in this study contains no parameters or adjustments that are specific to melanoma morphology, suggesting it can be used for analyzing other tissues and phenotypes, as well as potentially different image modalities and contrast techniques.
Melanoma progression; histopathological image analysis; non-parametric image analysis; H&E data; tissue classification
Flow cytometry specializes in high content measurements of cells and particles in suspension. Having long excelled in analytical throughput of single cells and particles, only recently with the advent of HyperCyt sampling technology has flow cytometry’s multi-experiment throughput begun to approach the point of practicality for efficiently analyzing hundreds-of-thousands of samples, the realm of high throughput screening (HTS). To extend performance and automation compatibility we built a HyperCyt-linked Cluster Cytometer platform, a network of flow cytometers for analyzing samples displayed in high-density, 1536-well plate format. To assess performance we used cell and microsphere based HTS assays that had been well characterized in previous studies. Experiments addressed important technical issues: challenges of small wells (assay volumes 10 μL or less, reagent mixing, cell and particle suspension), detecting and correcting for differences in performance of individual flow cytometers, and the ability to reanalyze a plate in the event of problems encountered during the primary analysis. Boosting sample throughput an additional four-fold, this platform is uniquely positioned to synergize with expanding suspension array and cell barcoding technologies in which as many as 100 experiments are performed in a single well or sample. As high-performance flow cytometers shrink in cost and size, cluster cytometry promises to become a practical, productive approach for HTS and other large scale investigations of biological complexity.
Flow cytometry; suspension array; high content analysis; high throughput screening
Reporter genes may serve as endogenous contrast agents in the field of photoacoustic (PA) molecular imaging (PMI), enabling greater characterization of detailed cellular processes and disease progression. To demonstrate the feasibility of using ferritin as a reporter gene, human melanoma SK-24 (SK-MEL-24) cells were co-transfected with plasmid expressing human heavy chain ferritin (H-FT) and plasmid expressing enhanced green fluorescent protein (pEGFP-C1) using lipofectamine™ 2000. Non-transfected SK-MEL-24 cells served as a negative control. Fluorescent imaging of GFP confirmed transfection and transgene expression in co-transfected cells. To detect iron accumulation due to ferritin overexpression in SK-MEL-24 cells, a focused high-frequency ultrasonic transducer (60 MHz, f/1.5), synchronized to a pulsed laser (fluence < 5 mJ/cm2) was used to scan the PA signal at a wide range NIR wavelengths (850–950 nm). PA signal intensity from H-FT transfected SK-MEL-24 cells was about 5–9 dB higher than nontransfected SK-MEL-24 cells at 850–950 nm. Immunofluorescence and RT-PCR analysis both indicate high levels of ferritin expression in H-FT transfected SK-MEL24 cells, with little ferritin expression in nontransfected SK-MEL-24 cells. In this study, the feasibility of using ferritin as a reporter gene for PMI has been demonstrated in vitro. The use of ferritin as a reporter gene represents a novel concept for PMI using an endogenous contrast agent and may provide various opportunities for molecular imaging and basic science research.
photoacoustics; molecular imaging; photoacoustic molecular imaging; endogenous contrast agent; reporter gene; ferritin
The complex life cycle of Plasmodium falciparum (Pf) makes it difficult to limit infections and reduce the risk of severe malaria. Improved understanding of Pf blood-stage growth and development would provide new opportunities to evaluate and interfere with successful completion of the parasite’s life cycle. Cultured blood stage Pf was incubated with Hoechst 33342 (HO) and thiazole orange (TO) to stain DNA and total nucleic acids, respectively. Correlated HO and TO fluorescence emissions were then measured by flow cytometry. Complex bivariate data patterns were analyzed by manual cluster gating to quantify parasite life cycle stages. The permutations of viable staining with both reagents were tested for optimal detection of parasitized RBC (pRBC). Pf cultures were exposed to HO and TO simultaneously to achieve optimal staining of pRBC and consistent quantification of early and late stages of the replicative cycle (rings through schizonts). Staining of Pf nucleic acids allows for analysis of parasite development in the absence of fixatives, lysis, or radioactivity to enable examination of erythrocytes from parasite invasion through schizont rupture using sensitive and rapid assay procedures. Investigation of the mechanisms by which anti-malarial drugs and antibodies act against different Pf lifecycle stages will be aided by this cytometric strategy.
flow cytometry; FACS; Hoechst 33342; thiazole orange; malaria; erythrocytes
T regulatory cells; polychromatic flow cytometry; HIV infection; phenotype; Tregs; OMIP
Amyloid beta (Aβ) oligomers and phosphorylated tau (p-tau) aggregates are increasingly identified as potential toxic intermediates in Alzheimer's disease (AD). In cortical AD synapses, p-tau co-localizes with Aβ, but the Aβ and p-tau peptide species responsible for synaptic dysfunction and demise remains unclear. The present experiments were designed to use high-speed cell sorting techniques to purify synaptosome population based on size, and then extend the method to physically isolate Aβ-positive synaptosomes with the goal of understanding the nature of Aβ and tau pathology in AD synapses. To examine the purity of size-gated synaptosomes, samples were first gated on size; particles with sizes between 0.5 and 1.5 microns were collected. Electron microscopy documented a homogenous population of spherical particles with internal vesicles and synaptic densities. Next, size-gated synaptosomes positive for Aβ were collected by fluorescence activated sorting and then analyzed by immunoblotting techniques. Sorted Aβ-positive synaptosomes were enriched for APP and for Aβ oligomers and aggregates; immunolabeling for p-tau showed a striking accumulation of p-tau aggregates compared to the original homogenate and purified synaptosomes. These results confirm co-localization of Aβ and p-tau within individual synaptic terminals and provide proof of concept for the utility of flow sorting synaptosomes.
synaptosome; flow cytometry; Alzheimer's disease; amyloid beta; p-tau
Extracellular hydrodynamic forces may be transmitted to the interior of cells through the alteration of integrin conformation and affinity. Integrin activation regulates leukocyte recruitment, cell activation, and transmigration. The cellular and molecular mechanisms for integrin activation are not precisely known, although intracellular calcium signaling is involved. Flow cytometry offers a versatile way to study intracellular calcium signaling in real-time. We report a novel method to generate defined shear by using a miniature Couette. Testing involved measuring shear induced intracellular calcium signals of human monoblastoid U937 cells in suspension. The Couette was connected externally to a flow cytometer and pressurized at 6 PSI (4.1 N/m2). Cells were subjected to well-defined shear between 0 and 1000 s−1 and delivered continuously within 10 s to a FACScan at 1 μl/s. Intracellular calcium levels and the percentage of cells activated increased as shear increased in duration and intensity.
Investigations of rare cell types in peripheral blood samples, such as tumor, fetal and endothelial cells, represent an emerging field with several potentially valuable medical applications. Peripheral blood is a particularly attractive body fluid for the detection of rare cells as its collection is minimally invasive and can be repeated throughout the course of the disease. Because the number of rare cells in mononuclear cells can be very low (1 in 10 million), a large number of cells must be quickly screened, which places demanding requirements on the screening technology. While enrichment technology has shown promise in managing metastatic disease, enrichment can cause distortions of cell morphology that limit pathological identification, and the enrichment targeting adds additional constraints that can affect sensitivity. Here we describe a new approach for detecting rare leukemia cells that does not require prior enrichment.
We have developed an immunocytochemical assay for identification of leukemia cells spiked in peripheral blood samples, and a high-speed scanning instrument with high numerical aperture and wide field of view to efficiently locate these cells in large sample sizes. A multiplex immunoassay with four biomarkers was used to uniquely identify the rare cells from leukocytes and labeling artifacts.
The cytometer preserves the cell morphology and accurately locates labeled rare cells for subsequent high resolution imaging. The sensitivity and specificity of the approach show promise for detection of a low number of leukemia cells in blood (1 in 10 million nucleated cells).
The method enables rapid location of rare circulating cells (25M cells/min), no specific enrichment step, and excellent imaging of cellular morphology with multiple immunofluorescent markers. The cell imaging is comparable to other imaging approaches such as laser scan cytometry and image flow cytometry, but the cell analysis rate is many orders of magnitude faster making this approach practical for detection of rare cells.
Rare cell; minimal residual disease; acute lymphoblastic leukemia; image cytometry
Observations of intracellular O-linked β-N-acetylglucosamine (O-GlcNAc) protein modification are primarily performed by Western blot or immunofluorescence microscopy. The goal of this study was to develop a flow cytometric-based assay for O-GlcNAc signaling and thus provide a more quantitative and rapid method to facilitate clinical analyses. Isolated peripheral blood neutrophils were stimulated with fMLF after adherence to glass cover slips. Cells in suspension were treated with either fMLF or PMA. Unstimulated cells served as controls. Neutrophils were fixed with formaldehyde and permeabilized with cold methanol before intracellular O-GlcNAc staining. Cells on cover slips were analyzed by fluorescence microscopy, and suspension cell data were acquired by flow cytometry. O-GlcNAc protein modification was increased following neutrophil stimulation with either 100 nM fMLF or 10 nM PMA. Increases were detected following either treatment using both flow cytometry and fluorescence microscopy. The time necessary for the completion of staining, data acquisition, and analysis was considerably less using flow cytometry. In addition, flow cytometry allows for the analysis of a substantially greater number of cells. Neutrophil protein modifications by O-GlcNAc are rapidly detected using flow cytometry and provide information similar to that observed using fluorescence microscopy.
neutrophil; O-GlcNAc; fMLF; PMA; intracellular protein; flow cytometry; immunofluorescence microscopy
The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase TrkB serve important regulatory roles for multiple aspects of the biology of neurons including cell death, survival, growth, differentiation, and plasticity. Regulation of the local availability of BDNF/TrkB at distinct subcellular domains such as soma, dendrites, axons, growth cones, nerve terminals, and spines appears to contribute to their specific functions. In view of the variance in size and shape of neurons and their compartments, previous quantitative studies of the BDNF/TrkB protein and mRNA lacked a robust normalization procedure. To overcome this problem, we have established methods that use immunofluorescence detection of α-tubulin as a normalization factor for the quantitative analysis of protein and mRNA in primary rat cortical and striatal neurons in culture. The efficacy of this approach is demonstrated by studying the dynamic distribution of proteins and mRNA at different growth stages or conditions. Treatment of cultured neurons with KCl resulted in increased levels of TrkB protein, reduced levels of BDNF mRNA (composite of multiple transcripts) and a slight reduction in BDNF protein levels in the dendrites from the cortex. The KCl treatment also lowered the percentage of BDNF and TrkB proteins in the soma indicative of protein transport. Finally, analysis of the rat cortical and striatal neurons demonstrated comparable or even higher levels of BDNF/TrkB protein and BDNF mRNA in the neurons from the striatum. Thus, in contrast to previous observations made in vivo, striatal neurons are capable of synthesizing BDNF mRNA when cultured in growth media in vitro. The analytical approach presented here provides a detailed understanding of BDNF/TrkB levels in response to a variety of neuronal activities. Our methods could be used broadly, including applications in cell and tissue cytometry, to yield accurate quantitative data of gene expression in cellular and subcellular contexts.
immunofluorescence; fluorescence in situ hybridization; brain-derived neurotrophic factor; BDNF; TrkB; α-tubulin; cortical neuron; striatal neuron