Fluorescence-activated Cell Sorting Method Research Articles

Genomic analysis of circulating tumor cells to evaluate predictive biomarkers.

38 Background: To estimate the association between molecular biomarkers detected in circulating tumor cells (CTC) and tumor sensitivity to treatment, robust assays are needed before qualification in prospective clinical trials. Methods: To address the limitations of the current FDA cleared technology, we focused on improving our ability to isolate more purified CTC populations based on fluorescence-activated cell sorting (FACS) to capture EpCAM+, CD45−, DAPI− cells from patients with castration-resistant prostate cancer (CRPC). Androgen receptor (AR) and genes frequently mutated in CRPC have been selected from the integrative genomic profiling at MSK. We optimized the RainDance microfluidic PCR followed by targeted sequencing in low number of cancer cells, before proceeding to clinical samples. Results: On blood samples from124 patients with progressive CRPC, FACS method isolates an average 100-fold more EpCAM+ events compared to the current FDA cleared CellSearch assay. By FACS, >10 or >50 events were isolated in 88% or 58% of patients, compared to 32% or 10% of patients by CellSearch, respectively. FACS isolated cells express prostate-specific mRNAs (PSA, AR, TMPRSS2-ERG), as detected by an analytically validated multiplex RT-PCR, indicating that these EpCAM+ events are bona fide CTC. For genomic profiling, sufficient high quality DNA was obtained from as little as 50 CTC, with a recovery rate of 89% from FACS sorted samples and adequate sequencing coverage, and 1:4 detection threshold in a heterogeneous cell population. Selected missense mutations in AR, PIK3CA and TP53 found in CTC but not in WBC from same patient are further analyzed. Conclusions: Molecular alterations in CTC can potentially serve as predictive markers of sensitivity and clinical outcomes as surrogate tissue in clinical practice. We established standard operating procedures for specimen processing, and confirmed the sequencing coverage and polymorphism detection thresholds in a heterogeneous cell population. In the context of available samples collected from patients enrolled on AR-targeted therapies, we will generate data to qualify CTC as biomarkers under the Oncology Biomarker Qualification Initiative. [Table: see text]

Lectins as a Tool for Detecting Neural Stem/Progenitor Cells in the Adult Mouse Brain

Glycoconjugates are biopolymers that are broadly distributed in the central nervous system, including the cell surface of neural stem cells or neural precursor cells (NSCs/NPCs). Glycoconjugates can be recognized by carbohydrate-binding proteins, lectins. Two lectins, Phaseolus vulgaris lectin agglutinin E-form (PHA-E4) and wheat germ agglutinin (WGA) have been reported to be useful in isolating NSCs/NPCs by fluorescence-activated cell sorting (FACS) or immunopanning methods. In this study, we analyzed the lectin-binding properties of NSCs/NPCs in two neurogenic regions of the adult mouse brain to determine whether PHA-E4 and WGA exhibit specific binding patterns on sections and whether there are other lectins presenting the binding pattern similar to those of PHA-E4 and WGA in lectin histochemistry. Among nine types of lectins, peanut agglutinin was localized to the white matter and four lectins bound to cells within the subventricular zone (SVZ) of the lateral ventricle. Lectin histochemistry combined with immunohistochemistry demonstrated that one lectin, Ricinus communis agglutinin, specifically detected type A neuronal precursors and that the remaining three lectins, Agaricus bisporus agglutinin (ABA), PHA-E4, and WGA, recognized type B NSCs and type C transient amplifying cells in the SVZ. These three lectins also recognized type 1 quiescent neural progenitors and type 2a amplifying neural progenitors in the subgranular layer of the dentate gyrus. Lectin histochemistry of the neurosphere culture also yielded similar results. These observations suggest that, in addition to PHA-E4 and WGA, ABA lectin may also be applicable in FACS or immunopanning for the isolation of NSCs/NPCs.

Assessment of the purity and characteristics of rat splenic T cells isolated by one-step discontinuous gradient of percoll

DEPAMEDE, N.S. 2010. Assessment of the purity and characteristics of rat splenic T cells isolated by one-step discontinuous gradient of percoll. JITV 15(2): 157-164. T cells isolated from spleen lymphocytes of rat (Rattus rattus) have been commonly used in an immunological research. However the T cells are still contaminated with other splenic cells. Therefore a simple and reliable technique to isolate the T cells in one step process is needed. The aim of the present study was to assess whether T cells isolated from spleen lymphocytes of rat as a model by means of discontinuous density gradient of Percoll could give pure T cells with their activities as needed in an immunological research. Isolation was carried out in 5 density gradients of Percoll i.e. 1.052, 1.063, 1.075, 1.085, and 1.122 g/ml. The purity of the cell fractions was determined by immunocytochemistry and fluorescence-activated cell sorting (FACS) methods. The activity of each lymphocyte fraction was determined through cell proliferation assays by culturing the cells in the presence of phytohemaglutinin (PHA) and Concanavalin A (Con A) as T cells inducers. The FACS results show that fraction I and II were found to contain mostly B cells and macrophages. The fractions were obtained on the layers of gradient 1.052/1.063 and 1.063/1.075. Fraction III (gradient 1.075/1.085) contain T cells (43.2%) which consisted of CD4 (34.4%) and CD8 (18.6%), B cells (36.5%), and macrophages (10.0%). Fraction IV was strongly expressed T cell receptors (76.2%) which consisted of CD4 (63.6%) and CD 8 (20.0%), while B cells were found 20.0% and macrophages just about 0.6%. The activity of T cells as expressed by their respond to PHA and Con A shown that fraction III gave the most positive response, while fraction IV which contained the most ‘pure’ T cells gave less activity. From the present study it can be concluded that the discontinuous density gradient of Percoll method can be used to isolate and purify T cells in one step process. Furthermore, the present study indicated that T cells to proliferate in response to mitogen require other immune cells such as B cells and macrophages.

CD133 Expression Defines a Tumor Initiating Cell Population in Primary Human Ovarian Cancer

Evidence is accumulating that solid tumors contain a rare phenotypically distinct population of cells, termed cancer stem cells (CSC), which give rise to and maintain the bulk of the tumor. These CSC are thought to be resistant to current chemotherapeutic strategies due to their intrinsic stem-like properties and thus may provide the principal driving force behind recurrent tumor growth. Given the high frequency of recurrent metastasis associated with human ovarian cancer, we sought to determine whether primary human ovarian tumors contain populations of cells with enhanced tumor-initiating capacity, a characteristic of CSC. Using an in vivo serial transplantation model, we show that primary uncultured human ovarian tumors can be reliably propagated in NOD/SCID mice, generating heterogeneous tumors that maintain the histological integrity of the parental tumor. The observed frequency of tumor engraftment suggests only certain subpopulations of ovarian tumor cells have the capacity to recapitulate tumor growth. Further profiling of human ovarian tumors for expression of candidate CSC surface markers indicated consistent expression of CD133. To determine whether CD133 expression could define a tumor-initiating cell population in primary human ovarian tumors, fluorescence-activated cell sorting (FACS) methods were employed. Injection of sorted CD133(+) and CD133(-) cell populations into NOD/SCID mice established that tumor-derived CD133(+) cells have an increased tumorigenic capacity and are capable of recapitulating the original heterogeneous tumor. Our data indicate that CD133 expression defines a NOD/SCID tumor initiating subpopulation of cells in human ovarian cancer that may be an important target for new chemotherapeutic strategies aimed at eliminating ovarian cancer.

The Isolation of Reticulocyte-Free Human Red Blood Cells

We depleted reticulocytes from erythrocytes of both sickle cell disease (SCD) subjects and healthy controls by four methods: fluorescence-activated cell sorting (FACS), Miltenyi immunomagnetic depletion (MACS), a combination of these methods (FACS + MACS) and Percoll density separation. The efficiency of these methods was assessed by new methylene blue staining and manual enumeration of the reticulocytes. FACS sorted erythrocytes from reticulocytes based on size and granularity, as well as the absence of dsDNA staining. MACS depleted reticulocytes from erythrocytes based on the immunoaffinity to CD36 and CD71. Reticulocytes from healthy controls were depleted to <or=0.1% using either the FACS or MACS method (alpha = 0.1). Reticulocytes from SCD subjects were depleted from 13.6% +/- 0.52% to 5.45% +/- 0.33% using MACS (n = 2), and from 10.9% +/- 0.47% to 2.0% +/- 0.2% using FACS (n = 4, alpha = 0.05). When combining FACS with MACS (n=3), the percentage of reticulocytes was decreased in SCD samples from 13.0% +/- 0.51% down to 1.5% +/- 0.17% (alpha = 0.1). Sedimentation through 75% percoll resulted in control and SCD samples being reduced from 0.27% +/- 0.6 (control) and 6.93% +/- 0.8 (SCD) reticulocytes to < 4.8 reticulocytes per million control RBCs and <2.5 per million SCD RBCs. This same method results in <2.1 leukocytes per million control RBCs and <3.7 per million SCD RBCs. We conclude that the percoll density method described here is the most effective method for isolating RBCs for proteomic analysis.

Isolation of Mouse Pancreatic Ductal Progenitor Cells Expressing CD133 and c-Met by Flow Cytometric Cell Sorting

Islet transplantation has become available across the globe since a novel protocol was reported. However, because donors are in short supply, only a minority of patients benefit from this procedure. Pancreatic progenitor cells are a promising resource for regeneration of new islets, but whether progenitor cells reside in ductal epithelium is not clear. Mouse pancreas was examined by immunohistochemistry with cell surface markers specific for ductal cells. We developed an isolation method for ductal cells by flow cytometric cell sorting using a newly identified specific marker for ductal cells. By using an in vitro colony assay, we characterized their proliferative and multipotent capacity. CD133 is expressed specifically in ductal epithelium. Flow cytometric analysis revealed that purified ductal cells are highly enriched in the CD133(+)CD34(-)CD45(-)Ter119(-) fraction. An analysis of clonal epithelial colonies formed by individual cells revealed that progenitor cells with multilineage differentiation capacity are present in neonatal ductal epithelium. Moreover, these progenitor cells express c-Met. In adult mice, progenitor cells that show a high proliferative capacity but appear committed to a ductal lineage are co-purified with CD133(+)CD34(-)CD45(-)Ter119(-) cells. We established a system for isolating and culturing mouse pancreatic ductal cells that relies on flow cytometric cell sorting. Clonal analysis revealed that a population of progenitor cells is present among CD133(+) ductal cells. Isolation of these cells will facilitate future studies into the roles of pancreatic progenitor cells in regeneration and carcinogenesis.

Enrichment of osteogenic cell populations from rat bone marrow stroma

The presence of multiple cell types in bone marrow and their varying proportions from isolation to isolation may count for the considerable variation in the outcome of different experiments. The presence of these multiple subpopulations suggests a need for a method that can purify the osteogenic component, i.e. osteoprogenitors, from other components. The availabilities of monoclonal antibodies recognizing subpopulations of osteoblasts are providing means for antibody-based methods. The cell surface antigens STRO-1, ALP and HOP-26 were used for cell sorting experiments with fluorescence activated cell sorting (FACS). These cell populations were analyzed on differential gene expression, cell proliferation and differentiation into the osteoblastic lineage. The oligo-microarray results showed that only the ALP positive cell population expressed genes of the extracellular matrix; like different collagens, ECM-1 and matrix protease MMP-14. The real-time polymerase chain reaction (QPCR) results showed that STRO-1 and ALP positive cells had an upregulation in expression of lipoprotein lipase, osteocalcin, and collagen type I. Integrin beta-3 was only upregulated for ALP positive cells, while for these cells downregulation occurred for the genes myosine, alkaline phosphatase and integrin beta-1. HOP-26 positive cells showed an upregulation in collagen type I compared to control group. The DNA analysis revealed that the cells of the control group and the HOP-26 positive cells showed a 5 times higher cell growth compared to the STRO-1 and ALP positive cells. The alkaline phosphatase activity showed no activity for the control group. The STRO-1 and ALP positive cells had a higher activity compared to the HOP-26 positive. The calcium measurements revealed only for the control group calcium at day 24. Based on the results of our study, we conclude that the FACS method had no negative effect on the proliferating as well as differentiating response of the cells. Further, we conclude that by using an antibody-based cell selection method, different cell populations with different mRNA expression profiles and different osteogenic characteristics can be obtained.

Combined inhibition of the phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways results in synergistic effects in glioblastoma cells

The present study uses cell-based screening assays to assess the anticancer effects of targeting phosphatidylinositol 3-kinase-regulated integrin-linked kinase (ILK) in combination with small-molecule inhibitors of Raf-1 or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK). The objective was to determine if synergistic interactions are achievable through the use of agents targeting two key cell signaling pathways involved in regulating glioblastoma cancer. The phosphatidylinositol 3-kinase/protein kinase B (PKB)/Akt and the Ras/MAPK pathway were targeted for their involvement in cell survival and cell proliferation, respectively. The glioblastoma cell lines U87MG, SF-188, and U251MG were transiently transfected with an antisense oligonucleotide targeting ILK (ILKAS) alone or in combination with the Raf-1 inhibitor GW5074 or with the MEK inhibitor U0126. Dose and combination effects were analyzed by the Chou and Talalay median-effect method and indicated that combinations targeting ILK with either Raf-1 or MEK resulted in a synergistic interaction. Glioblastoma cells transfected with ILKAS exhibited reduced levels of ILK and phosphorylated PKB/Akt on Ser473 but not PKB/Akt on Thr308 as shown by immunoblot analysis. These results were confirmed using glioblastoma cells transfected with ILK small interfering RNA, which also suggested enhanced gene silencing when used in combination with U0126. U87MG glioblastoma cells showed a 90% (P < 0.05) reduction in colony formation in soft agar with exposure to ILKAS in combination with GW5074 compared with control colonies. A substantial increase in Annexin V-positive cells as determined by using fluorescence-activated cell sorting methods were seen in combinations that included ILKAS. Combinations targeting ILK and components of the Ras/MAPK pathway result in synergy and could potentially be more effective against glioblastoma cancer than monotherapy.

Growth Hormone Enhances Natural Killer Cell Activity Against Glioma

Although it is now known that pituitary hormones activate the immune system, there are still numerous questions to be answered with regard to the mechanisms involved. In this study, which focuses on growth hormone (GH) and natural killer (NK) cells, the latter's antitumor effects on glioma were investigated. Using fluorescein isothiocyanate-labeled rat 9L glioma and NK-receptive YAC-1 cells as target cells and rat splenocytes as effector cells, a cytotoxicity assay was carried out with the fluorescence-activated cell sorter method, which stains dead cells with propidium iodide. The effector cells were pretreated 48 hours in advance with various concentrations of GH. A similar experiment was also carried out in the presence of anti-asialo-GM1 antibodies. When the GH concentration of 9L was 10 to 40 microg/ml, cytotoxicity was confirmed to have been enhanced 17% to 39%. This enhancement disappeared in the presence of anti-asialo-GM1 antibodies. A similar increase in cytotoxic activity was also confirmed in YAC-1 cells. In this experiment we observed GH enhancement of natural killer activity against glioma.

Expression of Melan-A/MART-1 in primary melanoma cell cultures has prognostic implication in metastatic melanoma patients.

The lack of melanoma-associated antigen (MAA) expression has been associated with the reduced overall survival in melanoma patients. In order to investigate whether the MAA expression detected on cell cultures established from melanoma patients might relate to the overall survival in these patients, we screened primary cell cultures derived from 37 melanoma metastases for the expression of five known MAA: Melan-A, tyrosinase, gp-100, MAGE-1 and MAGE-3 by polymerase chain reaction (PCR) and fluorescence-activated cell sorting (FACS). MAA expression detected by PCR was found at a high percentage in evaluated melanoma cell lines: 25 of 28 (89%) were positive for Melan-A, 22 of 28 (79%) were positive for tyrosinase, 26 of 28 (93%) were positive for gp-100, and 18 of 28 (64%) were positive for MAGE-3 expression. Using the FACS method the percentage of MAA-positive cell lines was much lower: 14 of 31 (45%) cell lines were positive for Melan-A, eight of 31 (26%) were positive for tyrosinase, 13 of 31 (42%) were positive for gp-100, six of 31 (19%) were positive for MAGE-1, and 14 of 31 (45%) were positive for MAGE-3 expression. Kaplan-Meier survival analysis demonstrated that the patients whose cell lines were positive for Melan-A expression by PCR had significantly longer overall survival time as Melan-A PCR-negative cases (P=0.0038). This could not be shown for any of the markers tested by FACS. Our results suggest that the expression of Melan-A/MART-1 in patient-derived cell cultures may help to identify a group of melanoma patients with prolonged survival.

New approaches to fluorescence compensation and visualization of FACS data

The Fluorescence Activated Cell Sorter (FACS) is an invaluable tool for clinicians and researchers alike in phenotyping and sorting individual cells. With the advances in FACS methodology, notably intracellular staining for cytokines, transcription factors and phosphoproteins, and with increases in the number of fluorescence detection channels, researchers now have the opportunity to study individual cells in far greater detail than previously possible. In this chapter, we discuss High-Definition (Hi-D) FACS methods that can improve analysis of lymphocyte subsets in mouse and man. We focus on the reasons why fluorescence compensation, which is necessary to correct for spectral overlap between two or more fluorochromes used in the same staining combination, is best done as a computed transformation rather than using the analog circuitry available on many flow cytometers. In addition, we introduce a new data visualization method that scales the axes on histograms and two-dimensional contour (or dot) plots to enable visualization of signals from all cells, including those that have minimal fluorescence values and are not properly represented with traditional logarithmic axes. This “Logicle” visualization method, we show, provides superior representations of compensated data and makes correctly compensated data look correct. Finally, we discuss controls that facilitate recognition of boundaries between positive and negative subsets.

Identification of B-cell subsets: an exposition of 11-color (Hi-D) FACS methods.

In the last few years, the effectiveness of developmental and functional studies of individual subsets of cells has increased dramatically owing to the identification of additional subset markers and the extension of fluorescence-activated cell sorter (FACS) capabilities to simultaneously measure the expression of more markers on individual cells. For example, introduction of a 6-8 multiparameter FACS instrument resulted in significant advances in understanding B-cell development. In this chapter, we describe 11-color high-dimensional (Hi-D) FACS staining and data analysis methods that provide greater clarity in identifying the B-cell subsets in bone marrow, spleen, and peritoneal cavity. Further, we show how a single Hi-D FACS antibody reagent combination is sufficient to unambiguously identify most of the currently defined B-cell developmental subsets in the bone marrow (Hardy fractions A-F) and the functional B-cell subsets (B-1a, B-1b, B-2, and marginal zone [MZ] B cells) in the periphery. Although we focus on murine B-cell subsets, the methods we discuss are relevant to FACS studies conducted with all types of cells and other FACS instruments. We introduce a new method for scaling axes for histograms or contour plots of FACS data. This method, which we refer to as Logicle visualization, is particularly useful in promoting correct interpretations of fluorescence-compensated FACS data and visual confirmation of correct compensation values. In addition, it facilitates discrimination of valid subsets. Application of Logicle visualization tools in the Hi-D FACS studies discussed here creates a strong new base for in-depth analysis of B-cell development and function.

Development of a multidose formulation for a humanized monoclonal antibody using experimental design techniques.

The purpose of this study was to identify optimal preservatives for a multidose formulation of a humanized monoclonal antibody using experimental design techniques. The effect of antimicrobial parenteral preservatives (benzyl alcohol, chlorobutanol, methylparaben, propylparaben, phenol, and m-cresol) on protein stability was assessed using size-exclusion chromatography, differential scanning calorimetry, right-angle light scattering, UV spectroscopy, and potency testing using a cell-based fluorescence-activated cell sorting method. A quick, cost-effective preservative screening test was designed. Combinations of preservatives were examined using an I-optimal experimental design. The protein was most stable in the presence of methylparaben and propylparaben, and was compatible with benzyl alcohol and chlorobutanol at low concentrations. Phenol and m-cresol were not compatible with the protein. The I-optimal experimental design indicated that as an individual preservative, benzyl alcohol was promising. The model also indicated several effective combinations of preservatives that satisfied the antimicrobial efficacy and physical stability constraints. The preservative screening test and the experimental design approach were effective in identifying optimal concentrations of antimicrobial preservatives for a multidose protein formulation; (1) benzyl alcohol, and (2) the combination of methylparaben and chlorobutanol were screened as potential candidates to satisfy the regulatory requirements of various preservative efficacy tests.

Lymphocyte surface thiol levels.

Recent studies have implicated reduced thiols (cysteine -SH) in the function of individual cell surface proteins. Studies presented here demonstrate that the overall level of reduced thiols on cell surface molecules differs on individual subsets of peripheral blood mononuclear cells and that these levels can be manipulated in vitro by altering the level of intracellular glutathione (iGSH). To quantitate cell surface thiols, we have developed a Hi-D (11-color) fluorescence-activated cell sorter method in which we covalently couple a fluorescent molecule, Alexa-maleimide, to free (reduced) -SH groups on proteins or other molecules exposed on the cell surface (exofacial membrane). In addition, to reveal changes in cell surface thiol levels in response to various in vitro treatments, we used a pair of fluorescent Alexa dyes with distinct excitation and emission spectra to stain the cells before and after treatments. These in vitro studies demonstrate that decreasing iGSH, by specifically inhibiting its synthesis, decreases cell surface molecule thiols (csm-SH) and that preventing loss of iGSH also prevents loss of csm-SH. However, examination of peripheral blood mononuclear cell subsets tested immediately after isolation from healthy or HIV-infected subjects failed to reveal a similar relationship between internal iGSH and csm-SH. Although there is a relatively wide variation between individuals in both csm-SH and iGSH, there is no correlation between median iGSH and csm-SH compared for 22 healthy and 36 HIV-infected subjects. Collectively, our findings indicate that local environment plays a greater role in determining the redox status of cell surface molecules than the internal redox status of the cells.

Fluorescent flow cytometric assay: a new diagnostic tool for measuring β-glucocerebrosidase activity in Gaucher disease

The purpose of this study was to determine glucocerebrosidase activity by a fluorescent flow cytometry method in patients and carriers of Gaucher disease, and in healthy controls, and correlate the results with the standard glucocerebrosidase assay in the same individuals. Biochemical diagnosis has heretofore been performed by measuring enzyme activity using a fluorimetric assay method with cell lysate. We present the results of a quantitative fluorescence-activated cell sorter (FACS) assay for diagnosis of Gaucher disease. Twenty-eight patients, 15 obligate carriers, and 21 healthy controls were tested by both the standard and FACS methods. Fluorescent products were obtained by intracellular hydrolysis of fluorescein di-β-glucopyranoside and measured by fluorescent flow cytometer. Activity was then expressed as an index ratio of mean fluorescence of the patient sample relative to control. Specificity of the assay for lysosomal β-glucocerebrosidase was demonstrated using conditurol-β-epoxide (CBE), a specific irreversible inhibitor of β-glucocerebrosidase. Both methods were performed on blood samples without knowledge of the genetic results. The mean ± SD results using FACS were: controls 1.12 ± 0.26, patients with Gaucher disease 0.21 ± 0.09 and carriers 0.76 ± 0.15. Using the standard method, mean enzyme activities were: controls 35.7 ± 9.0 uU/mg protein, patients with Gaucher disease 8.4 ± 4.5 uU/mg protein, and carriers 32.2 ± 6.9 uU/mg protein. Thus, FACS is a reliable and easy to use method for determination of enzyme activity in Gaucher disease, with excellent correlation with the standard method. The FACS method allows single cell enzyme evaluation rather than global activity of cell lysate, and gives excellent separation between patients, carriers, and controls.

A potential role for cx43-hemichannels in staurosporin-induced apoptosis.

To address the role of gap junction hemichannels in apoptosis, the cell death induced by staurosporine (ST) was evaluated in wild type HeLa cells (HeLa-WT) and transfectants expressing either full-length connexin43 (HeLa-Cx43) or a C-terminal truncation of Cx43 (HeLa-DeltaCT). Cell death was measured with fluorescence-activated cell sorting (FACS), both DNA and nuclear fragmentation methods and assays for PARP and caspase 3. The ST-mediated cell death was accelerated in HeLa-Cx43 cells compared to HeLa-WT and HeLa-DeltaCT. To determine why HeLa-Cx43 cells were more susceptible to ST, the phosphorylation state and the localization of Cx43 protein within cells were examined using specific Cx43 antibodies. The phosphorylated forms of Cx43 were sharply reduced in HeLa-Cx43 cells treated with ST. Moreover, in ST-treated HeLa-Cx43 cells, Cx43 was mainly observed at the cell surface. In contrast, the truncated form of Cx43 found in HeLa-DeltaCT cells, which lacks many of the normal phosphorylation sites, was observed in the cytosol with ST treatment. To examine the hemichannels in the plasma membranes of ST-treated HeLa-Cx43 cells, several dye uptake methods using carboxyfluorescein and propidium iodide were employed. While the number of fluorescent cells did not change in HeLa-WT and HeLa-DeltaCT cells with ST treatment, the number of fluorescent HeLa-Cx43 cells increased more than ten-fold. These results indicate that the increases in cell surface Cx43 seen with immunofluorescence and the elevated hemichannel activities detected with dye uptake could help explain the accelerated cell death observed in ST-treated HeLa-Cx43 cells.

The History and Future of the Fluorescence Activated Cell Sorter and Flow Cytometry: A View from Stanford

The Fluorescence Activated Cell Sorter (FACS) was invented in the late 1960s by Bonner, Sweet, Hulett, Herzenberg, and others to do flow cytometry and cell sorting of viable cells. Becton Dickinson Immunocytometry Systems introduced the commercial machines in the early 1970s, using the Stanford patent and expertise supplied by the Herzenberg Laboratory and a Becton Dickinson engineering group under Bernie Shoor. Over the years, we have increased the number of measured FACS dimensions (parameters) and the speed of sorting to where we now simultaneously measure 12 fluorescent colors plus 2 scatter parameters. In this history, I illustrate the great utility of this state-of-the-art instrument, which allows us to simultaneously stain, analyze, and then sort cells from small samples of human blood cells from AIDS patients, infants, stem cell transplant patients, and others. I also illustrate analysis and sorting of multiple subpopulations of lymphocytes by use of 8-12 colors. In addition, I review single cell sorting used to clone and analyze hybridomas and discuss other applications of FACS developed over the past 30 years, as well as give our ideas on the future of FACS. These ideas are currently being implemented in new programs using the internet for data storage and analysis as well as developing new fluorochromes, e.g., green fluorescent protein and tandem dyes, with applications in such areas as apoptosis, gene expression, cytokine expression, cell biochemistry, redox regulation, and AIDS. Finally, I describe new FACS methods for measuring activated kinases and phosphatases and redox active enzymes in individual cells simultaneously with cell surface phenotyping. Thus, key functions can be studied in various subsets of cells without the need for prior sorting.

Intracellular delivery strategies for antisense phosphorodiamidate morpholino oligomers.

Antisense oligonucleotides inhibit gene expression by interfering with transcription, translation, or splicing. They show great potential as gene-specific, nontoxic therapy for a wide variety of diseases. They are also powerful tools to study gene function as well as for validation of therapeutic targets. Even with compelling evidence of activity in vivo, the majority of cell types in culture require technologies capable of efficiently delivering antisense oligonucleotides into the cytosolic/nuclear compartment of the cells in culture. Phosphorodiamidate morpholino oligomers (PMO) are a new generation of antisense oligomers with high specificity and efficacy. They inhibit translation of targeted mRNA by steric blockade. Different methods were evaluated for efficient delivery of PMO into the cells in culture. Efficacy was compared using the PMO targeted to the 5'-untranslated region (5'-UTR) of alpha-globin-luciferase reporter fusion gene mRNA. A functional assay based on the lunciferase reporter system was used to measure efficacy. The fluorescence-activated cell sorting (FACS) method was used for quantitative determination of PMO uptake into the cells. Physical methods, such as scrape-loading, syringe-loading, and osmotic-loading, provided efficient transfer of PMO into the cells, which resulted in higher efficacy. These procedures caused minimal damage to the cells. Cell permeabilization with streptolysin O did not improve the cell uptake of PMO. Complexation with cationic lipids, Lipofectin and Lipofectamine (GIBCO-BRL, Gaithersburg, MD) also failed to enhance the uptake of PMO. We conclude that physical methods are optimal for the delivery of neutrally charged PMO into cells in culture. Further, these methods do not leave residual material that may interfere with the interpretation of targeted gene function.

CNTF and GDNF, but not NT-4, support corticospinal motor neuron growth via direct mechanisms.

Axotomy and neurodegenerative diseases cause corticospinal motor neuron (CSMN) degeneration. We previously showed that CNTF, NT-4 and GDNF can support CSMN survival in enriched preparations. Here we developed a fluorescence-activated cell sorting method to highly purify CSMN (97+/-4.6%). We tested the neurotrophic activities of CNTF, NT-4 and GDNF on enriched and purified CSMN preparations. Similar to their effects on enriched CSMN preparations, CNTF and GDNF sustained the survival of purified CSMN for at least 5 days with ED50 values of 1.28+/-0.46 nM and 0.59+/-0.39 nM, respectively. In contrast, NT-4 supported survival of enriched but not of purified CSMN, indicating that CNTF and GDNF sustain motor neuron survival by direct action of CSMN, while NT-4 requires accessory cells present in enriched CSMN preparations.

A sensitive method for quantifying cytomegalic endothelial cells in peripheral blood from cytomegalovirus-infected patients.

A sensitive method has been developed for the quantification of cytomegalic endothelial cells (CEC) in peripheral blood (PB) of patients with active cytomegalovirus (CMV) infections. The three subsequent key steps of this method are density centrifugation to enrich endothelial cells (EC) in the mononuclear cell (MNC) fraction, EC-specific staining, and fluorescence-activated cell sorting (FACS) of EC onto adhesion slides. The FACS method was compared with the conventional method of cytocentrifugation of the MNC fraction onto slides, followed by EC-specific staining. The main advantage of the additional steps for the isolation and quantification of CEC in PB by FACS is a 10-times-greater sensitivity than by cytocentrifugation of the MNC fraction alone. The recovery percentages of EC from whole blood were comparable for both methods. Recoveries of EC obtained after FACS were 53% +/- 16.5%, (mean +/- standard deviation), and recoveries of EC obtained after cytocentrifugation of the MNC fraction were 43% +/- 4.3%. In patients with active CMV infection, 5 to 72 CEC were detected by FACS, equivalent to 0.8 to 9.0 CEC/ml of blood. With this method for isolation and quantification, the characterization of CEC in PB of patients with CMV-associated clinical symptoms, as well as the quantification of EC in PB of patients with pathophysiological manifestations involving endothelial damage that are different from those caused by CMV infections, can be performed.