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Results for “Flow Cytometry”

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PubMed1997

Identification and analysis of macrophage-derived foam cells from human atherosclerotic lesions by using a "mock" FL3 channel in flow cytometry.

Macrophages are one of the major cell types in atherosclerotic lesions. They are believed to play an important role in the pathogenesis and development of the lesion, but their functional state and phenotypic characteristics are not well understood. Using flow cytometry, we analyzed surface markers of macrophages extracted from tissue digests. However, conventional techniques were hampered by the abundance of cell debris and extracellular lipids, which co-localized with double-positive cells in all fluorescent plots. We therefore developed a method to overcome this problem by using a novel gating technique in multiparameter flow cytometry. This method utilized the third fluorescence channel (FL3) as a "mock" channel, since no antibody conjugated to an FL3-specific fluorochrome was added to the samples. Cells single-positive for macrophage-specific monoclonal antibodies (mAb) conjugated to phycoerythrin (PE) (FL2) were separated from non-specific fluorescent particles in the FL2 versus FL3 fluorescent plot and a region excluding debris could be set. This was then used as a gate to exclude debris also in the first fluorescence channel (FL1) vs. FL2 plot in which expression of a panel of activation markers identified by fluorescein isothiocyanate (FITC)-conjugated mAb was analyzed. Using this strategy, we were able to identify and analyze the phenotype of macrophages from human atherosclerotic lesions. We were also able to sort these cells and in this way obtained a preparation of macrophage-derived foam cells from the tissue with little contamination of debris.

Antigens, Surface↗

PubMed1992

The use of ATP bioluminescence assay and flow cytometry in predicting radiosensitivity of uterine cancer cell lines: correlation of radiotoxicity and cell cycle kinetics.

Radiotherapy remains an important part of uterine cancer treatment. This study was designed to evaluate the potential of the ATP bioluminescence assay and flow cytometry for predicting radiosensitivity. Correlation of these two modalities revealed important insights into the relationship of radiotoxicity and cell kinetic effects. Six human uterine cancer cell lines were used: AE7, ECC1, HEC1A, HEC1B, AN3, and SKUT1B. Doses of cobalt 60 were 0, 1, 2, 5, 8, and 10 Gy. The ATP bioluminescence assays were performed on Day 7. Cell samples were taken at 0, 24, 48, 72, 96, and 168 hr for flow cytometry. The linear-quadratic model was used to fit survival data and mean inactivation dose D was calculated. Among parameters such as D, alpha and beta coefficients, and surviving fraction at 2 Gy (SF2), both D and SF2 correlated best with survival data. Radiation effects on the cell cycle did not correlate with D and revealed two distinct patterns: either a G1 accumulation with mild G2 block or a G1 depletion and severe G2 block. The S cells consistently demonstrated a biphasic pattern with an initial reduction followed by an accumulation. In summary, the ATP assay was shown to have potential in the study of radiosensitivity. Radiation-induced cell kinetics appeared to vary with intrinsic cellular differences and, thus, could not be used to predict radiosensitivity.

Adenosine Triphosphate↗

PubMed1998

Utilization of fine-needle aspiration cytology and flow cytometry in the diagnosis and subclassification of primary and recurrent lymphoma.

BACKGROUND: The primary diagnosis of non-Hodgkin's lymphoma/leukemia (NHL) by fine-needle aspiration (FNA) is controversial. The authors reviewed their experience with FNA and flow cytometry (FC) to determine the usefulness and limitations of these techniques in the diagnosis of NHL. METHODS: Slides and reports from all lymph node and extranodal FNAs performed during the period July 1993 to January 1997 with a diagnosis of lymphoma or benign lymphoid process were reviewed. Clinical and biopsy follow-up data were recorded. Results were tabulated and the usefulness of cytology was analyzed. RESULTS: There were 100 adequate aspirates from 87 patients. These included 72 cases of NHL, 58 (80%) of which were diagnosed by FNA and FC without the need for histologic sampling (69% of the primary lymphomas and 88% of the recurrent lymphomas). There were 22 aspirates suspicious for lymphoma, 12 equivocal results, and 7 benign diagnoses. Eighty-six percent of malignant FNAs (50 of 58) had flow cytometry (FC) as compared with only 15% (5 of 34) of the suspicious or equivocal FNAs. CONCLUSIONS: FNA is a valuable method for diagnosing and subclassifying NHL, although immunophenotyping by FC is often an essential ancillary test. In our experience, correlating the FNA results with the FC results can eliminate the need for a more invasive surgical biopsy in many cases.

Biopsy, Needle↗

PubMedDate not supplied

In vitro determination of the apoptotic effect of heparin on lymphoblasts using DNA analysis and measurements of Fas and Bcl-2 proteins by flow cytometry.

Heparin has an apoptotic effect beside its anticoagulant, anti-inflammatory, antihypertensive, and antiproliferative effects. In this study, the authors detected the percentages of apoptotic lymphoblasts and the expressions of apoptotic Fas protein and antiapoptotic Bcl-2 protein with flow cytometry in vitro after the incubation of lymphoblasts with heparin. Eleven newly diagnosed acute lymphoblastic leukemia (ALL) children were included in the study. Lymphoblasts were incubated in all different levels of heparin concentrations (0, 10, and 20 U/mL) and the percentages of apoptotic lymphoblasts and the percentages of Fas protein and Bcl-2 proteins were simultaneously measured by flow cytometry at 0, 1, and 2 h. At 0, 1, and 2 h, apoptosis was determined when heparin was added in 10- and 20-U/mL concentrations (p <.05). The apoptotic effect of heparin on lymphoblasts was higher at the first hour than at 0 and 2 h in 10- and 20-U/mL heparin concentrations (p <.01). The highest apoptosis was detected in the 20-U/mL heparin concentration at the first hour. The expression levels of Fas protein on lymphoblasts were higher at the first hour than at 0 and 2 h in 10- and 20-U/mL heparin concentrations (p <.001). The highest expression of Fas protein was observed in the 20-U/mL heparin concentration at the first hour. The expression levels of Bcl-2 protein on lymphoblasts were lower at the first hour than at 0 and 2 h in 10- and 20-U/mL heparin concentrations (p <.001). The lowest expression of Bcl-2 protein was detected in the 20-U/mL heparin concentration at the first hour. Increased concentrations of heparin had an increasing effect on the percentages of apoptotic lymphoblasts. The expression percentages of Fas protein on lymphoblasts also increased, whereas the expression percentages of Bcl-2 protein on lymphoblasts decreased (p <.05). These results suggest that low-dose heparin may cause significant apoptosis of lymphoblasts in newly diagnosed ALL patients.

Apoptosis↗

PubMed2000

Simultaneous measurement of cellular P-glycoprotein content and function by multiparametric flow-cytometry.

OBJECTIVE: A multiparametric approach was applied to simultaneously determine expression and function of the drug efflux pump P-glycoprotein (PGP) in multidrug-resistant (MDR) human leukemic lymphoblast cell lines and isolated leukemic blasts using flow-cytometry in a patient with acute myeloid leukemia (AML). METHODS: The antigen was measured by staining PGP using the monoclonal antibody 4e3 which does not inhibit the function of PGP. The 4e3 antibody binds to an external epitope of PGP and can therefore be used for staining living cells. Drug transport, mediated by PGP, was determined simultaneously by measuring rhodamine 123 (rho123) efflux. The MDR cell lines, CEM/VLB10-2 and CEM/VBL100 are 10-fold and 270-fold resistant to vinblastine (VBL), respectively, compared to the human PGP-negative parent cell line CEM/WT and they express different amounts of PGP. Initially, living cells were stained using the 4e3 antibody and a secondary antibody labeled with 7-amino-4-methylcoumarin-3-acetic acid (AMCA). Cells were then incubated for 60 min with rho123 (10 microM) and analyzed for rhodamine and AMCA-derived fluorescence. The decrease in rho123 fluorescence was determined after a further period of 30 min. RESULTS: CEM/VLB100 cells expressed larger amounts of PGP, and rho123 fluorescence after 30 min was 85% lower than the parent cell line. PGP expression and rho123 efflux were also detected in CEM/VLB10-2 cells which display a low degree of resistance, thus reflecting the high sensitivity of this method. PGP-expressing blasts and moderate rho123 efflux were also observed in a specimen derived from a patient with clinically resistant acute myeloid leukemia (AML). CONCLUSION: A multiparametric approach using flow-cytometry allows the reliable and sensitive measurement of both PGP expression and function simultaneously in single cells.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2006

Quantification of cell fusion by flow cytometry.

Cells of different types can be induced to fuse by electroshock. Cells of one type are typically dominant and are able to reprogram the nuclei derived from cells of the other type, in fusion hybrids derived from one cell of each type. Flow cytometry provides a quick and objective technique to assess cell fusion for nuclear reprogramming studies. Two cell types are each stained with a different fluorescent dye and then induced to fuse to form fusion products called heterokaryons. Heterokaryons can be identified and quantified by flow cytometry as double-stained events. Protocols are provided for the optimization of cell staining under conditions that minimize cell clumping and dye leakage. If spectral overlap occurs between emission spectra of the two stained cell types, the data will need to be electronically compensated.

Animals↗

PubMed1992

Nuclear planimetry and DNA flow cytometry of verruga peruana.

Verruga peruana is a vascular hyperplasia that is sometimes histologically mistaken for a malignant neoplasm. An infiltrative pattern with nuclear pleomorphism and mitoses is prominent in some lesions. From a series of lesions of verruga peruana, we selected five atypical lesions, two of them closely resembling epithelioid angiosarcoma. We used histologic criteria as well as morphometry and flow cytometry to further characterize the atypical features of the lesions. Histology and morphometry evidenced marked nuclear pleomorphism. Flow cytometry failed to reveal aneuploidy and disclosed about 10% of the cells to be in the S, G2 and M phase.

Bartonella Infections↗

PubMed1991

DNA flow cytometry in sperm cells from testicular cancer patients. Impact of different treatment modalities on spermatogenesis.

In 77 patients with testicular cancer, sperm cell density was evaluated before and after treatment together with the following parameters as recorded by DNA flow cytometry: the percentage of condensed and non-condensed haploid sperm cells, and the number of non-haploid cells. The patients received either abdominal radiotherapy, cisplatin-based combination chemotherapy (+/- surgery) or did not undergo antiproliferative treatment at all. Ejaculates with a low sperm cell density had high numbers of non-condensed haploid and non-haploid cells, whereas the percentage of condensed haploid cells was decreased. However, even 'azoospermic' semen samples (by light microscopy) contained haploid cells indicating ongoing sperm cell production. One year after radiotherapy there was a significant decrease in the sperm cell density and the number of condensed haploid cells. Chemotherapy led to a significant reduction of sperm cell density evaluated 1 year after treatment. Three years after the diagnosis of testicular cancer, sperm cell production had recovered in most patients. In patients who did not have radiotherapy or chemotherapy the median density of sperm cells 3 years after treatment significantly exceeded the corresponding figure from the pretreatment situation. A low pretreatment percentage of condensed haploid cells (less than or equal to 90%) was correlated with the lack of 1-year recovery of sperm cell production. It is too early to state whether longitudinal flow cytometric data from ejaculates in testicular cancer patients yield clinically valuable information, in addition to that obtained by light microscopy. From this preliminary observation it seems that DNA flow cytometry in ejaculates from testicular cancer patients may give valuable clinical information about sperm cell production following treatment.

Antineoplastic Agents↗

PubMed1989

General principles of multiparameter flow cytometric analysis: applications of flow cytometry in the diagnostic pathology laboratory.

Recent developments in the design of flow cytometric instrumentation and advances in computerized methods of data analysis have produced flow cytometers that are compatible with the clinical laboratory setting. Because flow cytometry is impacting on virtually every area of diagnostic pathology (particularly the subspecialties of hematology, immunology, infectious disease, transplantation monitoring, neoplasia, and genetics), it is essential that pathologists develop a general understanding of flow cytometric techniques. In addition to new developments in instrumentation, simplification of laser design and advances in fluorochrome chemistry have provided several new sophisticated tools to make multiple correlative biological measurements on normal and neoplastic cells in the clinical setting. Flow cytometric analysis thus allows the pathologist to more effectively diagnose, predict prognosis, and monitor disease progression. This report reviews the basic components of flow cytometric instrumentation, the physical and biological parameters that may be assessed using flow cytometric techniques, and the multiparametric data analysis systems available for the clinical laboratory.

Antibodies, Monoclonal↗

PubMed1989

Use of biocarrier beads and flow cytometry for single-cell studies of fibronectin gene regulation in dibutyryl cyclic AMP reverse transformed CHO-K1 cells.

The protease sensitivity of a number of cell surface or cytoskeletal components and the relationship of these to the substratum in attached cells has prevented the quantitative measurement of their expression by flow cytometry. Using traditional cell sorting techniques, cells must be treated with a protease to detach them from a substrate in order to produce a single-cell suspension. Unfortunately, proteolytic treatment alters or destroys a number of cellular proteins. Fibronectin either on the cell surface or as part of the substratum laid down by the cell is particularly sensitive to proteases, preventing its quantitative study by flow cytometry. To circumvent these problems and produce a single cell suspension necessary for flow cytometric analysis, CHO-K1, a Chinese hamster ovary cell line, were grown in suspension on specially-treated 25 microns biocarrier beads. The CHO-K1 cell line is composed of transformed epithelial-like cells that have lost the fibronectin deposit around their cell membranes. To restore the typical fibroblastic deposit of fibronectin, the cells attached to beads were induced by dibutyryl cAMP to undergo a reverse transformation reaction to restore fibroblastic morphology and the typical fibroblastic deposite of fibronectin on the cell surface and substratum. The cells attached to beads were then immunofluorescently labeled for the protease-sensitive, extracellular matrix component, fibronectin, and examined on a flow cytometer. Cell surface fibronectin heterogeneity was then examined on a cell-by-cell basis as a function of cell cycle using Hoechst 33342 dye that binds to AT base pairs of cellular DNA. Dual laser measurement and multiparameter list mode data analysis were used to study the relationship between cell surface fibronectin of biocarrier bead attached cells and cell cycle.

Animals↗

PubMed1996

Rapid detection of IgG subclasses on DAT positive RBC membranes by flow cytometry (FC).

UNLABELLED: Red blood cell (RBC)-bound IgG can cause hemolysis resulting, e.g. in severe cases of autoimmune hemolytic anemias (AIHA) or in hemolytic disease of the newborn (HDN). Serologic detection and differentiation of these antibodies are often difficult in cases of low antibody titers. We investigated 36 cases of poly- and monospecific IgG-positive DATs by flow cytometry. The RBC samples were washed, diluted, and incubated with monoclonal antibodies directed against IgG1, IgG2, IgG3, and IgG4, respectively. Analysis was performed on a flow cytometer. Nine cases were negative for all 4 IgG subclasses, 8 cases were positive for IgG2, 5 for IgG1, 5 for IgG3, and 3 for all four subclasses. In 6 patients we found combinations of 2 or 3 subclasses (2 for IgG1 and IgG2, 1 for IgG1 and IgG3, 1 for IgG3 and IgG4, 1 for IgG1, IgG2 and IgG4, 1 for IgG1, IgG3 and IgG4). Serological differentiation revealed specific anti-bodies only in 3 cases (anti-Lea, anti-Leb, anti-P1). The type of IgG subclass and the amount of RBC-bound IgG is relevant for the degree of in vivo RBC destruction. Flow cytometry provides a rapid, highly sensitive, cost efficient, and specific tool for IgG detection, including subgroups. CONCLUSIONS: We therefore recommend flow cytometric analysis to be integrated into the serological decision process as an additional method for serological problem cases.

Adult↗

PubMed2004

Quantification of cell hybridoma yields with confocal microscopy and flow cytometry.

The fusion of antigen presenting and cancer cells leads to the formation of hybrid cells, which are considered a potential vaccine for treating cancer. The quality assessment of hybrid cell vaccines is crucial for the introduction of this new treatment. Flow cytometry was the method used recently, since it is faster in comparison to classical microscopy. Here we describe a rapid confocal microscopy based approach to quantify hybrid cell yields. The extent of fusion rate was determined by confocal microscopy by counting dual fluorescent cells and by measuring the area of co-localized pixels. Results of both methods showed high degree of correlation. The same samples were also analyzed by flow cytometry. Fusion rates determined with both techniques showed significant correlation. In conclusion, using confocal microscopy we developed a sensitive and a rapid method to assess the yield of hybridomas in a large number of electrofused cells.

Animals↗

PubMed1998

Purification of human fetal hippocampal neurons by flow cytometry for transplantation.

We have established primary cultures, highly enriched in neurons, from the hippocampus of human fetal brains at 20-23 gestational weeks. More than 80% of cells were viable when seeded. Neurons were isolated from primary cultures by flow cytometry to a high degree of purity, as demonstrated by immunocytochemical staining. FACS scanning analysis using a DNA-staining dye showed that hippocampal neurons did not divide in culture. To demonstrate that FACS-sorted neurons can be transplanted and integrated into the host brain, neuron-enriched primary culture from human fetal striatum was infected with a viral-mediated vector containing a reporter gene, beta-galactosidase. Striatal neurons were subsequently purified by flow cytometry and transplanted into the striatum of rats. Following transplantation, the rat brains were processed for beta-galactosidase histochemistry and electron microscopy. Beta-galactosidase expression indicates that transplanted human neurons survived in the host and were metabolically active. The transplanted neurons received synaptic inputs, as judged from the presence of presynaptic terminals on their surface. Our study demonstrates connectivity between transplanted human fetal primary neurons and host tissue at the ultrastructural level. Our results support the feasibility of ultimately transplanting neurons into humans as a possible treatment for recovery of the nervous system (e.g., neurodegenerative diseases).

Animals↗

PubMed2007

A relational database for management of flow cytometry and ELISpot clinical trial data.

BACKGROUND: Although relational databases are widely used in bioinformatics with deposited and finalized data, they have not received widespread usage among immunologists for managing raw laboratory data such as that generated by ELISpot or flow cytometry assays. Almost no published guidance exists for immunologists to design appropriate and useful data management systems. METHODS: We describe the design and implementation of a Microsoft Access relational database used in a clinical trial in which the primary immunogenicity measures were ELISpot and intracellular cytokine staining. RESULTS: Our data management system enabled us to perform sophisticated queries and to interpret our data as quantitatively as possible. It could easily be used without modification by other researchers using automated plate reading of ELISpot plates or four color flow cytometry. CONCLUSION: We illustrate in detail the use of a flexible data management system for two of the most widely used immunological techniques. Minor modifications for more colors or other outputs can easily be implemented. Based on this example, other modifications could be easily envisaged for any other quantitative output.

Biological Specimen Banks↗

PubMed1987

In vitro proliferation of primary human head and neck squamous cell carcinomas evaluated by flow cytometry.

In vitro proliferation of primary human head and neck squamous cell carcinomas was investigated using single cell suspensions and tissue explants of primary specimens and xenografts from 20 tumor specimens. The evaluations of the cells emerging in culture were performed with flow cytometry. Epithelial-like cells proliferated in serum-free medium, while no fibroblast-like cells were observed in culture. The epithelial-like cells could be subcultured several passages before senescence occurred. Conditioned medium or serum supplementation was necessary for a sustained outgrowth of malignant squamous cells as documented by flow cytometry. From a tumor line established in nude mice slowly proliferating tumor cells emerged. After 4-5 months in culture tumor cells seemed to be adapted to the culture conditions used. This resulted in a more consistent tumor cell proliferation. Early passage cultures from primary human head and neck squamous cell carcinomas are clearly difficult to obtain either from primary human specimens or from tumor lines established in nude mice.

Animals↗

PubMed2001

Flow cytometry and the study of central nervous disease in patients with acute leukaemia.

Central nervous system (CNS) leukaemia is still a matter of debate and new technologies are required to improve the classic morphological definition. One hundred and sixty-eight cerebrospinal fluid (CSF) samples from 31 patients with acute leukaemia were analysed by flow cytometry and conventional cytology. Concordant positive and negative findings were found in 158 samples but 10 produced discrepant results. Cytology seemed to offer more precise information in one CSF sample and flow cytometric accuracy could be demonstrated in five samples. We conclude that flow cytometry is of great help in confirming CNS leukaemia and eliminating other conditions. Therefore, leukaemic patients can benefit from double cytological and flow cytometric CSF studies.

Acute Disease↗

PubMed2000

Susceptibility of human herpesvirus 6 to antiviral compounds by flow cytometry analysis.

BACKGROUND: The emergence of human herpesvirus 6 (HHV-6) as a human pathogen led to the possibility of specific therapy against HHV-6 and the development of standardized susceptibility assays of HHV-6 to antivirals. METHODS: We have developed a flow cytometry method to analyze the multiplication of the HST strain of human herpesvirus 6 (HHV-6) variant B in vitro using monoclonal antibodies specific to virus proteins. This method was subsequently used to determine the sensitivity of HST multiplication in MT4 cells to four antiviral compounds of three different classes: acyclovir (ACV) and ganciclovir (GCV), two acyclic guanosine analogs; cedofovir (CDV), an acyclic nucleoside phosphonate; and phosphonoformic acid (PFA), a pyrophosphate analog. RESULTS: The 50% inhibitory concentrations (IC(50)) of ACV, GCV, CDV, and PFA determined by flow cytometry assay were 25.3, 6.4, 0.95, and 6.0 microM, respectively (5.7, 1.6, 0.3, and 1.8 microg/ml, respectively). These data together with the results of cytotoxicity assays confirmed the high efficiency and selectivity of CDV and PFA against HHV-6 B in vitro, suggested by previous results. CONCLUSIONS: Our flow cytometric assay appeared as a reproducible specific method to characterize HHV-6 susceptibility to antiviral compounds. It can be considered as a convenient alternative to the other immunologic and DNA hybridization assays used for that purpose.

Antibodies, Monoclonal↗

PubMed1991

DNA flow cytometry of large-bowel biopsies showing that adenoma tissue predicts the ultimate nature of the lesions.

Ploidy of neoplastic large-bowel cells in lesions from which biopsies showed only adenoma tissue by histopathologic examination was investigated retrospectively by DNA flow cytometry. Biopsy and resected-specimen blocks gave interpretable ploidy profiles from 83 lesions. The expectation that adenomas will show diploidy in biopsies and that adenocarcinomas will show aneuploidy in biopsies was fulfilled in 80% of the lesions. In addition, four lesions with diploid biopsies were polyps with minimal invasion. Thus, diploidy would have correctly predicted that local resection was adequate treatment for 62 lesions and conversely that cancer resection was the appropriate treatment for 8 lesions, for an overall predictive success rate of 87%. In conjunction with endoscopic, radiologic and other data, DNA flow cytometry of biopsies from apparently benign large-bowel lesions could become a useful addition to therapeutic decision-making.

Adenocarcinoma↗