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1.  [No title available] 
PMCID: PMC3907470  PMID: 23900967
2.  [No title available] 
PMCID: PMC4293101  PMID: 23281001
3.  Relationship Between DNA Damage Response, Initiated by Camptothecin or Oxidative Stress, and DNA Replication, Analyzed by Quantitative 3D Image Analysis 
A method of quantitative analysis of spatial (3D) relationship between discrete nuclear events detected by confocal microscopy is described and applied in analysis of a dependence between sites of DNA damage signaling (γH2AX foci) and DNA replication (EdU incorporation) in cells subjected to treatments with camptothecin (Cpt) or hydrogen peroxide (H2O2). Cpt induces γH2AX foci, likely reporting formation of DNA double-strand breaks (DSBs), almost exclusively at sites of DNA replication. This finding is consistent with the known mechanism of induction of DSBs by DNA topoisomerase I (topo1) inhibitors at the sites of collisions of the moving replication forks with topo1-DNA “cleavable complexes” stabilized by Cpt. Whereas an increased level of H2AX histone phosphorylation is seen in S-phase of cells subjected to H2O2, only a minor proportion of γH2AX foci coincide with DNA replication sites. Thus, the increased level of H2AX phosphorylation induced by H2O2 is not a direct consequence of formation of DNA lesions at the sites of moving DNA replication forks. These data suggest that oxidative stress induced by H2O2 and formation of the primary H2O2-induced lesions (8–oxo-7,8-dihydroguanosine) inhibits replication globally and triggers formation of γH2AX at various distances from replication forks. Quantitative analysis of a frequency of DNA replication sites and γH2AX foci suggests also that stalling of replicating forks by Cpt leads to activation of new DNA replication origins.
PMCID: PMC3888650  PMID: 23846844
DNA damage; DNA damage response; DDR; γH2AX; histone H2AX phosphorylation; DNA replication; camptothecin; oxidative stress; hydrogen peroxide; cell cycle; base excision repair
4.  The effect of cell subset isolation method on gene expression in leukocytes 
Multiple scientific disciplines require the isolation of specific subsets of blood cells from patient samples for gene expression analysis by microarray or RNA-sequencing, preserving disease- or treatment-related signatures. However, little is known with respect to the impact of different cell isolation methods on gene expression and the effects of positive selection, negative selection and fluorescence activated cell sorting (FACS) have not previously been assessed in parallel. To address this knowledge gap, CD4+ T cells, CD8+ T cells, B cells and monocytes were isolated from blood samples from 5 independent donors using positive immunomagnetic selection, negative immunomagnetic selection and FACS. We hypothesized that positive selection and FACS would yield higher purity but may have an impact on gene expression since both methods utilize antibodies that bind surface receptors of the cell type of interest. Moreover, FACS might upregulate stress response genes due to passage of the cells through the sorter. Microarray gene expression data was generated and subjected to unsupervised clustering and differential gene expression analysis. Surprisingly, these analyses revealed that gene expression signatures were more similar between cells isolated by negative selection and FACS compared to cells isolated by positive selection. Moreover, genes that are involved in the response to stress generally had the highest expression in cells isolated by negative or positive selection and not FACS. Thus, FACS is the recommended method for isolation of leukocyte subsets for gene expression studies since this method results in the purest subset populations and does not appear to induce a stress response.
PMCID: PMC3975050  PMID: 24115734
negative immunomagnetic selection; positive immunomagnetic selection; fluorescent activated cell sorting; CD4+ T cell; CD8+ T cell; B cell; monocyte; gene expression; microarray
5.  A computer vision approach to rare cell in vivo fluorescence flow cytometry 
Non-invasive enumeration of rare circulating cell populations in small animals is of great importance in many areas of biomedical research. In this work we describe a macroscopic fluorescence imaging system and automated computer vision algorithm that allows in vivo detection, enumeration and tracking of circulating fluorescently-labeled cells from multiple large blood vessels in the ear of a mouse. This imaging system uses a 660 nm laser and a high sensitivity electron-multiplied charge coupled device camera (EMCCD) to acquire fluorescence image sequences from relatively large (~5 × 5 mm2) imaging areas. The primary technical challenge was developing an automated method for identifying and tracking rare cell events in image sequences with substantial autofluorescence and noise content. To achieve this, we developed a two-step image analysis algorithm that first identified cell candidates in individual frames, and then merged cell candidates into tracks by dynamic analysis of image sequences. The second step was critical since it allowed rejection of >97% of false positive cell counts. Overall, our computer vision IVFC (CV-IVFC) approach allows single-cell detection sensitivity at estimated concentrations of 20 cells per mL of peripheral blood. In addition to simple enumeration, the technique recovers the cell’s trajectory, which in the future could be used to automatically identify, for example, in vivo homing and docking events.
PMCID: PMC3934567  PMID: 24273157
In vivo flow cytometry; automated; rare cell; computer vision
6.  Nuclear translocation of Nuclear Factor of Activated T cells (NFAT) as a quantitative pharmacodynamic parameter for tacrolimus 
Nuclear Factor of Activated T cells (NFAT) is a family of transcription factors involved in regulating the immune response. The canonical NFAT pathway is calcium-dependent and upon activation, NFAT is dephosphorylated by the phosphatase, calcineurin. This results in its translocation from the cytoplasm to the nucleus and transcription of downstream target genes that include the cytokines IL-2, IL-10, and IFNγ. Calcineurin inhibitors including tacrolimus inhibit the NFAT pathway and are used as immunosuppressants in transplant settings to prevent graft rejection. There is, as yet, no direct means to monitor tacrolimus pharmacodynamics. In this study, a rapid, quantitative, image cytometry–based measurement of nuclear translocation of NFAT1 is used to evaluate NFAT activation in T cells and its tacrolimus-induced inhibition. A strong dose-dependent correlation between NFAT1 inhibition and tacrolimus dose is demonstrated in vitro. Time kinetic analysis of NFAT1 inhibition in plasma from stable renal transplant recipients before and after an in vivo dose with tacrolimus correlated with the expected pharmacokinetic profile of tacrolimus. This was further corroborated by analysis of patients' autologous CD4 and CD8 T cells. This is the first report to show that the measurement of NFAT1 activation potential by nuclear translocation can be used as a direct, sensitive, reproducible and quantitative pharmacodynamic readout for tacrolimus action. These results, and the rapid turnaround time for this assay, warrant its evaluation in a larger clinical setting to assess its role in therapeutic drug monitoring of calcineurin inhibitors.
PMCID: PMC4018210  PMID: 24136923
Nuclear Factor of Activated T Cells (NFAT1); Tacrolimus; Immunosuppression; Imaging Flow Cytometry
7.  Optimized flow cytometry isolation of murine spermatocytes 
Meiotic prophase I (MPI), is an initial stage of meiosis characterized by intricate homologous chromosome interactions, synapsis and DNA recombination. These processes depend on the complex, but poorly understood early MPI events of homologous chromosome search, alignment and pairing. Detailed molecular investigation of these early events requires isolation of individual MPI substages. Enrichment for Pachytene (P) and Diplotene (D) substages of late MPI was previously accomplished using flow cytometry. However, separation of early MPI spermatocytes, specifically, of Leptotene (L) and Zygotene (Z) substages, has been a challenge due to these cells’ similar characteristics. In this report, we describe an optimized Hoechst-33342 (Hoechst)-based flow cytometry approach for isolating individual MPI populations from adult murine testis. We get significant enrichment for individual L and Z spermatocytes, previously inseparable from each other, and optimize the isolation of other MPI substages. Our flow cytometry approach is a combination of three optimized strategies. The first is optimization of testis dissociation protocol that yields more consistent and reproducible testicular single cell suspension. The second involves optimization of flow cytometric gating protocol where a critical addition to the standard protocol for cell discrimination based on Hoechst fluorescence, involves a back-gating technique based on light scattering parameters. This step specifies selection of individual MPI substages. The third, is an addition of DNA content restriction to the gating protocol to minimize contamination from non-meiotic cells. Finally, we confirm significant enrichment of high-purity Preleptotene (PreL), L, Z, P and D MPI spermatocytes using stage-specific marker distribution. The technique will facilitate understanding of the molecular events underlying meiotic prophase I.
PMCID: PMC4246648  PMID: 24664803
Adult mouse; meiotic prophase I; Hoechst 33342; flow cytometry; cell sorting
8.  SWIFT—Scalable Clustering for Automated Identification of Rare Cell Populations in Large, High-Dimensional Flow Cytometry Datasets, Part 2: Biological Evaluation 
Cytometry  2014;85(5):422-433.
A multistage clustering and data processing method, SWIFT (detailed in a companion manuscript), has been developed to detect rare subpopulations in large, high-dimensional flow cytometry datasets. An iterative sampling procedure initially fits the data to multidimensional Gaussian distributions, then splitting and merging stages use a criterion of unimodality to optimize the detection of rare subpopulations, to converge on a consistent cluster number, and to describe non-Gaussian distributions. Probabilistic assignment of cells to clusters, visualization, and manipulation of clusters by their cluster medians, facilitate application of expert knowledge using standard flow cytometry programs. The dual problems of rigorously comparing similar complex samples, and enumerating absent or very rare cell subpopulations in negative controls, were solved by assigning cells in multiple samples to a cluster template derived from a single or combined sample. Comparison of antigen-stimulated and control human peripheral blood cell samples demonstrated that SWIFT could identify biologically significant subpopulations, such as rare cytokine-producing influenza-specific T cells. A sensitivity of better than one part per million was attained in very large samples. Results were highly consistent on biological replicates, yet the analysis was sensitive enough to show that multiple samples from the same subject were more similar than samples from different subjects. A companion manuscript (Part 1) details the algorithmic development of SWIFT. © 2014 The Authors. Published by Wiley Periodicals Inc.
PMCID: PMC4238823  PMID: 24532172
SWIFT; EM algorithm; flow cytometry clustering; ground truth data; automated analysis
9.  SWIFT—Scalable Clustering for Automated Identification of Rare Cell Populations in Large, High-Dimensional Flow Cytometry Datasets, Part 1: Algorithm Design 
Cytometry  2014;85(5):408-421.
We present a model-based clustering method, SWIFT (Scalable Weighted Iterative Flow-clustering Technique), for digesting high-dimensional large-sized datasets obtained via modern flow cytometry into more compact representations that are well-suited for further automated or manual analysis. Key attributes of the method include the following: (a) the analysis is conducted in the multidimensional space retaining the semantics of the data, (b) an iterative weighted sampling procedure is utilized to maintain modest computational complexity and to retain discrimination of extremely small subpopulations (hundreds of cells from datasets containing tens of millions), and (c) a splitting and merging procedure is incorporated in the algorithm to preserve distinguishability between biologically distinct populations, while still providing a significant compaction relative to the original data. This article presents a detailed algorithmic description of SWIFT, outlining the application-driven motivations for the different design choices, a discussion of computational complexity of the different steps, and results obtained with SWIFT for synthetic data and relatively simple experimental data that allow validation of the desirable attributes. A companion paper (Part 2) highlights the use of SWIFT, in combination with additional computational tools, for more challenging biological problems. © 2014 The Authors. Published by Wiley Periodicals Inc.
PMCID: PMC4238829  PMID: 24677621
automated multivariate clustering; rare subpopulation detection; Gaussian mixture models; weighted sampling; ground truth data
10.  OMIP-022: Comprehensive assessment of antigen-specific human T-cell functionality and memory 
Cytometry  2014;85(7):576-579.
PMCID: PMC4231567  PMID: 24866990
OMIP; cytometry; FACS; tuberculosis; T cell; ICS; cytokine staining; T-cell memory
11.  Diabetes Reduces Bone Marrow and Circulating Porcine Endothelial Progenitor Cells, an Effect Ameliorated by Atorvastatin and Independent of Cholesterol 
Bone marrow derived endothelial progenitor cells (EPCs) are early precursors of mature endothelial cells which replenish aging and damaged endothelial cells. The authors studied a diabetic swine model to determine if induction of DM adversely affects either bone marrow or circulating EPCs and whether a HMG-CoA reductase inhibitor (statin) improves development and recruitment of EPCs in the absence of cholesterol lowering. Streptozotocin was administered to Yorkshire pigs to induce DM. One month after induction, diabetic pigs were treated with atorvastatin (statin, n = 10), ezetimibe (n = 10) or untreated (n = 10) and evaluated for number of bone marrow and circulating EPCs and femoral artery endothelial function. There was no effect of either medication on cholesterol level. One month after induction of DM prior to administration of drugs, the number of bone marrow and circulating EPCs significantly decreased (P < 0.0001) compared to baseline. Three months after DM induction, the mean proportion of circulating EPCs significantly increased in the atorvastatin group, but not in the control or ezetimibe groups. The control group showed progressive reduction in percentage of flow mediated vasodilatation (no dilatation at 3 months) whereas the atorvastatin group and ezetimibe exhibited vasodilatation, 6% and 4% respectively. DM results in significant impairment of bone marrow and circulating EPCs as well as endothelial function. The effect is ameliorated, in part, by atorvastatin independent of its cholesterol lowering effect. These data suggest a model wherein accelerated atherosclerosis seen with DM may, in part, result from reduction in EPCs which may be ameliorated by treatment with a statin.
PMCID: PMC4221094  PMID: 19051240
diabetes mellitus; endothelium; endothelial progenitor cells; statin; inflammation; cytometry
12.  DNA Damage Signaling, Impairment of Cell Cycle Progression, and Apoptosis Triggered by 5-Ethynyl-2′-deoxyuridine Incorporated into DNA 
The “click chemistry” approach utilizing 5-ethynyl-2′-deoxyuridine (EdU) as a DNA precursor was recently introduced to assess DNA replication and adapted to flow- and imaging-cytometry. In the present study, we observed that EdU, once incorporated into DNA, induces DNA damage signaling (DDS) such as phosphorylation of ATM on Ser1981, of histone H2AX on Ser139, of p53 on Ser15, and of Chk2 on Thr68. It also perturbs progression of cells through the cell cycle and subsequently induces apoptosis. These effects were observed in non-small cell lung adenocarcinoma A549 as well as in B-cell human lymphoblastoid TK6 and WTK1 cells, differing in the status of p53 (wt versus mutated). After 1 h EdU pulse-labeling, the most affected was cells progression through the S phase subsequent to that at which they had incorporated EdU. This indicates that DNA replication using the template containing incorporated EdU is protracted and triggers DDS. Furthermore, progression of cells having DNA pulse-labeled with EdU led to accumulation of cells in G2, likely by activating G2 checkpoint. Consistent with the latter was activation of p53 and Chk2. Although a correlation was observed in A549 cells between the degree of EdU incorporation and the extent of γH2AX induction, such correlation was weak in TK6 and WTK1 cells. The degree of perturbation of the cell cycle kinetics by the incorporated EdU was different in the wt p53 TK6 cells as compared to their sister WTK1 cell line having mutated p53. The data are thus consistent with the role of p53 in modulating activation of cell cycle checkpoints in response to impaired DNA replication. The confocal microscopy analysis of the 3D images of cells exposed to EdU for 1 h pulse and then grown for 24 or 48 h revealed an increased number of colocalized γH2AX and p53BP1 foci considered to be markers of DNA double-strand breaks and enlarged nuclei.
PMCID: PMC3846616  PMID: 24115313
click chemistry; DNA strand breaks; p53 activation; Chk2 activation; ATM activation; γH2AX foci; p53BP1 foci; caspase-3 activation; laser scanning cytometry; flow cytometry; confocal microscopy; SBIP methodology
14.  Generalized Unmixing Model for Multispectral Flow Cytometry Utilizing Nonsquare Compensation Matrices 
Multispectral and hyperspectral flow cytometry (FC) instruments allow measurement of fluorescence or Raman spectra from single cells in flow. As with conventional FC, spectral overlap results in the measured signal in any given detector being a mixture of signals from multiple labels present in the analyzed cells. In contrast to traditional polychromatic FC, these devices utilize a number of detectors (or channels in multispectral detector arrays) that is larger than the number of labels, and no particular detector is a priori dedicated to the measurement of any particular label. This data-acquisition modality requires a rigorous study and understanding of signal formation as well as unmixing procedures that are employed to estimate labels abundance. The simplest extension of the traditional compensation procedure to multispectral data sets is equivalent to an ordinary least-square (LS) solution for estimating abundance of labels in individual cells. This process is identical to the technique employed for unmixing spectral data in various imaging fields. The present study shows that multispectral FC data violate key assumptions of the LS process, and use of the LS method may lead to unmixing artifacts, such as population distortion (spreading) and the presence of negative values in biomarker abundances. Various alternative unmixing techniques were investigated, including relative-error minimization and variance-stabilization transformations. The most promising results were obtained by performing unmixing using Poisson regression with an identity-link function within a generalized linear model framework. This formulation accounts for the presence of Poisson noise in the model of signal formation and subsequently leads to superior unmixing results, particularly for dim fluorescent populations. The proposed Poisson unmixing technique is demonstrated using simulated 8-channel, 2-fluorochrome data and real 32-channel, 6-fluorochrome data. The quality of unmixing is assessed by computing absolute and relative errors, as well as by calculating the symmetrized Kullback–Leibler divergence between known and approximated populations. These results are applicable to any flow-based system with more detectors than labels where Poisson noise is the dominant contributor to the overall system noise and highlight the fact that explicit incorporation of appropriate noise models is the key to accurately estimating the true label abundance on the cells.
PMCID: PMC4177998  PMID: 23526804
spectral unmixing; compensation; Poisson regression; multispectral and hyperspectral flow cytometry
15.  Pericytes: A Universal Adult Tissue Stem Cell? 
PMCID: PMC4165081  PMID: 22069300
multiparameter flow cytometry; perivascular cells; pericytes; adult tissue stem cells; supra adventitial-adipose stromal cells (SA-ASC)
16.  Flow Cytometric Determination of Stem/Progenitor Content in Epithelial Tissues: An Example from Nonsmall Lung Cancer and Normal Lung 
Single cell analysis and cell sorting has enabled the study of development, growth, differentiation, repair and maintenance of “liquid” tissues and their cancers. The application of these methods to solid tissues is equally promising, but several unique technical challenges must be addressed. This report illustrates the application of multidimensional flow cytometry to the identification of candidate stem/progenitor populations in non-small cell lung cancer and paired normal lung tissue. Seventeen paired tumor/normal lung samples were collected at the time of surgical excision and processed immediately. Tissues were mechanically and enzymatically dissociated into single cell suspension and stained with a panel of antibodies used for negative gating (CD45, CD14, CD33, glycophorin A), identification of epithelial cells (intracellular cytokeratin), and detection of stem/progenitor markers (CD44, CD90, CD117, CD133). DAPI was added to measure DNA content. Formalin fixed paraffin embedded tissue samples were stained with key markers (cytokeratin, CD117, DAPI) for immunofluorescent tissue localization of populations detected by flow cytometry. Disaggregated tumor and lung preparations contained a high proportion of events that would interfere with analysis, were they not eliminated by logical gating. We demonstrate how inclusion of doublets, events with hypodiploid DNA, and cytokeratin+ events also staining for hematopoietic markers reduces the ability to quantify epithelial cells and their precursors. Using the lung cancer/normal lung data set, we present an approach to multidimensional data analysis that consists of artifact removal, identification of classes of cells to be studied further (classifiers) and the measurement of outcome variables on these cell classes. The results of bivariate analysis show a striking similarity between the expression of stem/progenitor markers on lung tumor and adjacent tumor-free lung.
PMCID: PMC4162487  PMID: 23081669
non-small cell lung cancer; lung stem cells; flow cytometry data analysis; solid tissue; collagenase
17.  Mesenchymal markers on human adipose stem/progenitor cells 
The stromal-vascular fraction (SVF) of adipose tissue is a rich source of multipotent stem cells. We and others have described 3 major populations of stem/progenitor cells in this fraction, all closely associated with small blood vessels: endothelial progenitor cells (EPC, CD45−/CD31+/CD34+), pericytes (CD45−/CD31−/CD146+) and supra-adventitial adipose stromal cells (SA-ASC, CD45−/CD31−/CD146−/CD34+). EPC are luminal, pericytes are adventitial and SA-ASC surround the vessel like a sheath.
The multipotency of the pericytes and SA-ASC compartments is strikingly similar to that of CD45−/CD34−/CD73+/CD105+/CD90+ bone marrow-derived mesenchymal stem cells (BM-MSC). Here we determine the extent to which this mesenchymal expression pattern is expressed on the 3 adipose stem/progenitor populations. Eight independent adipose tissue samples were analyzed in a single tube (CD105-FITC/CD73-PE/CD146-PETXR/CD14-PECY5/CD33-PECY5/CD235A-PECY5/CD31-PECY7/CD90-APC/CD34-A700/CD45-APCCY7/DAPI).
Adipose EPC were highly proliferative with 14.3±2.8% (mean ± SEM) having >2N DNA. About half (53.1±7.6%) coexpressed CD73 and CD105, and 71.9±7.4% expressed CD90. Pericytes were less proliferative (8.2±3.4% >2N DNA) with a smaller proportion (29.6±6.9% CD73+/CD105+, 60.5±10.2% CD90+) expressing mesenchymal associated markers. However, the CD34+ subset of CD146+ pericytes, were both highly proliferative (15.1±3.6% with >2N DNA) and of uniform mesenchymal phenotype (93.3±3.7% CD73+/CD105+, 97.8±0.7% CD90+), suggesting transit amplifying progenitor cells. SA-ASC were the least proliferative (3.7 ± 0.8%>2N DNA) but were also highly mesenchymal in phenotype (94.4±3.2% CD73+/CD105+, 95.5±1.2% CD90+).
These data imply a progenitor/progeny relationship between pericytes and SA-ASC, the most mesenchymal of SVF cells. Despite phenotypic and functional similarities to BM-MSC, SA-ASC are distinguished by CD34 expression.
PMCID: PMC4157311  PMID: 23184564
multiparameter flow cytometry; adipose stromal vascular fraction; mesenchymal stem cells; endothelial progenitor cells; perivascular cells; pericytes; immunofluorescent microscopy; supra-adventitial adipose stromal cells
19.  Förster resonance energy transfer microscopy and spectroscopy for localizing protein-protein interactions in living cells 
The fundamental theory of Förster resonance energy transfer (FRET) was established in the 1940's. Its great power was only realized in the past 20 years after different techniques were developed and applied to biological experiments. This success was made possible by the availability of suitable fluorescent probes, advanced optics, detectors, microscopy instrumentation and analytical tools. Combined with state-of-the-art microscopy and spectroscopy, FRET imaging allows scientists to study a variety of phenomena that produce changes in molecular proximity, thereby leading to many significant findings in the life sciences. In this review, we outline various FRET imaging techniques and their strengths and limitations; we also provide a biological model to demonstrate how to investigate protein-protein interactions in living cells using both intensity- and fluorescence lifetime-based FRET microscopy methods.
PMCID: PMC3924896  PMID: 23813736
FRET; FLIM; FLIM-FRET; protein-protein interactions
20.  Statistical Classification of Multivariate Flow Cytometry Data Analyzed by Manual Gating: Stem, Progenitor, and Epithelial Marker Expression in Nonsmall Cell Lung Cancer and Normal Lung 
The use of supervised classification to extract markers from primary flow cytometry data is an emerging field that has made significant progress, spurred by the growing complexity of multidimensional flow cytometry. Whether the markers are extracted without supervision or by conventional gate and region methods, the number of candidate variables identified is typically larger than the number of specimens (p < n) and many variables are highly intercorrelated. Thus, comparison across groups or treatments to determine which markers are significant is challenging. Here, we utilized a data set in which 86 variables were created by conventional manual analysis of individual listmode data files, and compared the application of five multivariate classification methods to discern subtle differences between the stem/progenitor content of 35 non-small cell lung cancer and adjacent normal lung specimens. The methods compared include elastic-net, lasso, random forest, diagonal linear discriminant analysis, and best single variable (best-1). We described a broadly applicable methodology consisting of: (1) variable transformation and standardization; (2) visualization and assessment of correlation between variables; (3) selection of significant variables and modeling; and (4) characterization of the quality and stability of the model. The analysis yielded both validating results (tumors are aneuploid and have higher light scatter properties than normal lung), as well as leads that require followup: Cytokeratin+ CD133+ progenitors are present in normal lung but reduced in lung cancer; diploid (or pseudo-diploid) CD117+CD44+ cells are more prevalent in tumor. We anticipate that the methods described here will be broadly applicable to a variety of multidimensional cytometry problems.
PMCID: PMC4149906  PMID: 23239514
multivariate analysis; p < n problem; elastic net; lasso; random forest; diagonal linear discriminant analysis; nonsmall cell lung cancer; normal lung; stem cells
21.  Stromal vascular progenitors in adult human adipose tissue 
The in vivo progenitor of culture-expanded mesenchymal-like adipose-derived stem cells (ADSC) remains elusive, owing in part to the complex organization of stromal cells surrounding the small vessels, and the rapidity with which adipose stromal vascular cells adopt a mesenchymal phenotype in vitro.
Immunohistostaining of intact adipose tissue was used to identify 3 markers (CD31, CD34, CD146) which together unambiguously discriminate histologically distinct inner and outer rings of vessel-associated stromal cells, as well as capillary and small vessel endothelial cells. These markers were used in multiparameter flow cytometry in conjunction with stem/progenitor markers (CD90, CD117) to further characterize stromal vascular fraction (SVF) subpopulations. Two mesenchymal and two endothelial populations were isolated by high speed flow cytometric sorting, expanded in short term culture and tested for adipogenesis.
The inner layer of stromal cells in contact with small vessel endothelium (pericytes) was CD146+/α-SMA+/CD90±/CD34−/CD31−; the outer adventitial stromal ring (designated supra adventitial-adipose stromal cells, SA-ASC) was CD146−/α-SMA−/CD90+/CD34+/CD31−. Capillary endothelial cells were CD31+/CD34+/CD90+ (endothelial progenitor), while small vessel endothelium was CD31+/CD34−/CD90− (endothelial mature). Flow cytometry confirmed these expression patterns and revealed a CD146+/CD90+/CD34+/CD31− pericyte subset that may be transitional between pericytes and SA-ASC. Pericytes had the most potent adipogenic potential, followed by the more numerous SA-ASC. Endothelial populations had significantly reduced adipogenic potential compared to unsorted expanded SVF cells.
In adipose tissue perivascular stromal cells are organized in two discrete layers, the innermost consisting of CD146+/CD34− pericytes, and the outermost of CD146−/CD34+ SA-ASC, both of which have adipogenic potential in culture. A CD146+/CD34+ subset detected by flow cytometry at low frequency suggests a population transitional between pericytes and SA-ASC.
PMCID: PMC4148047  PMID: 19852056
multiparameter flow cytometry; adipose derived stem cells; adipose derived stromal cells; endothelial cells; perivascular cells; pericytes; immunofluorescence microscopy; supra adventitial-adipose stromal cells
22.  A novel method for monitoring tumor dormancy using fluorescent dye DiD 
Tumor dormancy is a condition in which tumor cells remain viable for a long period of time without significant growth, but retaining the potential to eventually regrow resulting in disease relapse after a long disease-free interval. Currently, due to limitations in existing avenues of study, little is known as to how tumor cells become dormant and how they leave dormancy to become full blown metastases. In this study, to explore the mechanisms of tumor dormancy, we used a lipophilic fluorescent dye, DiD, that rapidly and stably integrates into the phospholipid cell membrane. We cultured DiD-stained prostate cancer cell lines for 10 days and isolated cells by flow cytometry based on expression levels of DiD. We found that a decrease in DiD intensity was correlated to the reduction of EdU, where the DiD-high population proliferated more slowly than the DiD-low population and the DiD-low population exhibited a higher mitotic index. We also found that DiD was detected after 3 weeks of implantation in an in vivo setting. Importantly, DiD dye did not have any effect on normal cellular metabolism, including cell growth, migration, and apoptosis. Although further study is indicated, DiD can be useful for identifying the mechanisms underlying tumor dormancy.
PMCID: PMC4143457  PMID: 24700602
Tumor dormancy; DiD fluorescent dye; Prostate cancer; Proliferation; Flow Cytometry
23.  Laser Excitation Power and the Flow Cytometric Resolution of Complex Karyotypes 
The analytical resolution of individual chromosome peaks in the flow karyotype of cell lines is dependent on sample preparation and the detection sensitivity of the flow cytometer. We have investigated the effect of laser power on the resolution of chromosome peaks in cell lines with complex karyotypes. Chromosomes were prepared from a human gastric cancer cell line and a cell line from a patient with an abnormal phenotype using a modified polyamine isolation buffer. The stained chromosome suspensions were analyzed on a MoFlo sorter (Beckman Coulter) equipped with two water-cooled lasers (Coherent). A bivariate flow karyotype was obtained from each of the cell lines at various laser power settings and compared to a karyotype generated using laser power settings of 300 mW. The best separation of chromosome peaks was obtained with laser powers of 300 mW. This study demonstrates the requirement for high-laser powers for the accurate detection and purification of chromosomes, particularly from complex karyotypes, using a conventional flow cytometer.
PMCID: PMC4131141  PMID: 20506467
flow karyotype; resolution; chromosomes; laser power; photo-saturation
25.  International Society for the Advancement of Cytometry Cell Sorter Biosafety Standards 
Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99–117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414–437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment.
PMCID: PMC4117398  PMID: 24634405
flow cytometry; occupational health; biohazards; cell sorting; biosafety; aerosol containment

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