Cytometry最新文献

Background: Lipopolysaccharide (LPS) comprises the outer cell wall of all gram-negative bacteria. It consists of an oligosaccharide core and lipid A. All LPS-induced biological responses are lipid A-dependent. Once released, LPS triggers a host systemic inflammatory response that leads to septic shock. Binding studies have helped to reveal some of the molecular interactions behind septic shock. Such studies have employed methods of labeling bacterial LPS with either radiochemicals or fluorescent dyes. Poor labeling of the LPS has resulted in the use of high concentrations of LPS in order to detect its binding.

Methods: In this study, we have devised a new methodology for labeling LPS, using hydrazide and galactose oxidase in order to oxidize galactose residues to aldehyde groups in the oligosaccharide core of the LPS.

Results: We have managed to generate a conjugate that is highly fluorescent (LPS-to-Alexa 488 labeling ratio of 1:5) and biologically active.

Conclusions: For the first time, this probe has enabled us to detect LPS binding even at pg/ml concentrations. Using this methodology, any Alexa-hydrazide dye can be conjugated to LPS, providing us with novel probes for imaging studies.

Background: Increased mitochondrial generation of reactive oxygen intermediates (ROI) due to defective respiratory chain activity has been implicated in physiological processes such as apoptosis, in the pathogenesis of mitochondrial diseases, and as part of the normal aging process. Established methods addressing activity of the respiratory chain complexes have been limited to bulk assays for single parameters. This study describes a flow cytometry-based method and its validation for the detection of respiratory chain function in single cells permeabilized by digitonin.

Methods: Flow cytometry was used to measure mitochondrial membrane potential (DeltaPsi(m)) and reactive oxygen generation under differing conditions of respiration. This was brought about by the addition of substrates and inhibitors to digitonin-permeabilized cells. This method was validated by measurement of oxygen consumption and ATP production and by confocal microscopy.

Results: Activity of the respiratory chain complexes assessed by DeltaPsi(m) responded to substrates and inhibitors as predicted from assessment by oxygen consumption and ATP synthesis. In addition, the flow cytometry method allows the simultaneous assessment of mitochondrial ROI generation. This was confirmed by the localization of the ROI probe, carboxy-DCF, to the same site as the mitochondrial probe observed by confocal microscopy.

Conclusions: This method allows the functional integrity of the respiratory chain complexes to be studied at the single-cell level, thus addressing the relationship between disordered function of respiratory chain complexes and mitochondrial ROI generation.

Background: Megakaryocytes are classically identified by their cellular morphology and expression of platelet glycoproteins.

Methods: In this study, the expression of GPIIIa (CD61) on hemopoietic cells was analyzed by dual-fluorescence flow cytometry.

Results: All monocytic cells (CD14+) were shown to coexpress CD61. As the expression of platelet protein on these monocytic cells cannot be reduced by treating the cells with anticoagulant (ethlyenediaminetetraacetic acid [EDTA]), this observation is not simply due to platelet adhesion. When sorted CD61(lo)CD14+ cells were studied by light and electron microscopy, platelets or platelet fragments could not be detected on the cell surface. These cells were found to have typical monocytic morphology but no megakaryocytic characteristics.

Conclusions: This finding demonstrates that without careful definition, the quantitation of megakaryocytic cells will be inappropriately high. A clear and unambiguous criteria for the identification of megakaryocytic cells is described based on the high expression of platelet glycoprotein (e.g., CD61(hi) or CD41(hi)) but not the monocyte marker (CD14(neg)).

Background: Cryptosporidium is an important waterborne pathogen. Detection of Cryptosporidium in concentrated water samples depends on oocyst isolation using immunomagnetic separation (IMS) and/or fluorescence-activated cell sorting (FACS), followed by confirmation using immunofluorescence staining (IFA) and fluorescence microscopy. These methods require highly trained microscopists for oocyst identification and confirmation. Analysis is hampered due to the presence of autofluorescent particles coupled with particles binding nonspecifically with the monoclonal antibodies (mAbs) used for detection. Flow cytometry (FCM) has the potential to be a more specific method for oocyst detection, but such a system would require more than one selection parameter.

Methods: Various mAbs from commercial suppliers were paired with CRY104-PE and evaluated. The mAb combination that best discriminated stained oocyst from detritus was optimized and compared to Cryptosporidium detection utilizing one-color IFA/FACS.

Results: A highly specific two-color assay employing the IgG(1) mAb CRY104 was developed. The assay resulted in reductions, up to 20-fold, in the number of non-Cryptosporidium particles detected. The addition of a second selection parameter improved microscopic analysis times and simplified oocyst confirmation by microscopists.

Conclusions: A two-color assay employing competing surface mAbs reduces the number of fluorescent particles sorted, thus improving FCM detection methods for Cryptosporidium.

Background: Immunohistochemistry and immunofluorescence (IF) assays frequently rely on subjective observer evaluation for grading. The aim of our study was to develop an objective quantitative index based on confocal laser scanning microscopy (CLSM) and image analysis of an IF assay to determine alteration in protein expression levels in normal versus tumor tissue. The relative levels of Met expression, a prognostic factor in breast cancer, were used as a model for evaluating image analysis algorithms.

Methods: Primary human breast cancer biopsies were collected. Sections containing tumor and adjacent uninvolved normal regions were immunostained for Met and digital images were acquired by CLSM. Subsequently, the digital data were manipulated using several different algorithms to calculate prognostic indexes. The results were correlated with the clinical outcome to determine the prognostic value of these indexes.

Results: Different algorithms were used to obtain quantitative indexes to evaluate the relative levels of Met expression. We report a statistical correlation between patient prognosis and relative Met level in normal versus tumor tissue as determined by three distinct algorithms using Kaplan-Meier analysis (log-rank): calculations based on intensity levels differences DV (P = 0.002), DIntensity (P = 0.014), and entropy divergence (Dentropy; P = 0.0023).

Conclusions: Using adjacent normal tissue as an internal reference, a quantitative index of tumor Met level divergence can be objectively determined to have a prognostic value. Moreover, this methodology can be used for other proteins in a variety of different diseases.

Despite the existence of high interleukin (IL)-12 serum levels in patients with chronic active alcoholism, previous studies from our group have shown that, during active ethanol intake, alcoholic patients with alcoholic liver cirrhosis (ALC) display an impaired T-helper-1 response together with abnormalities in the peripheral blood (PB) cytotoxic compartment. The aim of the present study was to gain further insights into the mechanisms underlying these abnormalities. For that purpose, we analyzed the expression on PB B- and T-cell subsets of both the CD28 and CD80 costimulatory molecules, the ability of T lymphocytes to bind to exogenous recombinant IL-2, and the serum levels of soluble CD8 (sCD8) that might interfere with CD8+ T-cell activation in a group of 10 ALC patients with active ethanol intake (ALCET group). As reference groups, we analyzed 10 healthy individuals, 10 chronic alcoholic patients without liver disease (AWLD group) but with active ethanol intake, and 10 ALC patients who had quit drinking for at least 1 year. Our results showed that ALCET patients display a significant decrease in the number of PB CD28+/CD8(hi) T cells (P < 0.05) and CD80+ B cells (P < 0.01) compared with both healthy controls and AWLD patients. In addition, in ALCET patients, PB T cells also showed a decreased ability to bind to exogenous IL-2 (P < 0.01). This was associated with the existence of increased serum levels of sCD8 in ALC patients, the highest levels being detected in the ALCET group (P < 0.01). Altogether, our results point to the existence of several abnormalities that would affect the cytotoxic response in ALCET patients.

Although numerous studies of gastric cancers on DNA ploidy have been reported, differences in the degree of aneuploidy (DNA index, DI) during progression have not been identified. We attempted to chart the differences in DIs during progression to clarify the role of aneuploidy in gastric cancers. We classified the gastric cancers examined into intestinal (n = 88) and diffuse (n = 48) types, and then analyzed 136 gastric cancers (intramucosal cancer, 42; submucosal cancer, 39; advanced cancer, 55) by flow cytometry using multiple sampling. In addition, we examined the DNA ploidy pattern of mucosal and submucosal lesions using the same submucosal cancers to study the tumor progression in individual cancers. Intratumoral DNA differences in DNA ploidy were observed in both types of gastric cancers. In intestinal-type cancers, multiple subclones indicated by a different DI occurred during the early stage of gastric cancers, whereas in diffuse-type cancers, multiple subclones were found primarily in advanced cancers. Although the DI varied widely in early intestinal-type cancers between 1.0 and 2.0, in early diffuse-type cancers, the DI tended to be less than 1.2. However, in advanced stage gastric cancers, the DI distribution was similar for both histological types. In intestinal-type cancers, high DI (>1.3) aneuploidy was frequently found in mucosal lesions. In contrast, only low DI (<1.2) aneuploid clones were observed in mucosal lesions of diffuse-type cancers. The present results suggest that high DI aneuploid tumor clones in intramucosal cancers acquire invasive ability when they progress to submucosal cancers, whereas DNA aneuploidy itself plays an important role in submucosal invasion of diffuse-type cancers.

Background: During induction of apoptosis, the pro-apoptotic member of the Bcl-2 protein family (Bax) undergoes translocation to the mitochondria. The translocation, which leads to accumulation of Bax in the mitochondrial intermembrane space, appears to be the critical event determining release of cytochrome c to cytosol: the latter event triggers the irreversible steps of apoptosis, namely, the activation of caspases and the initiation of the degradation of many proteins. The aim of this study was to utilize the morphometric capabilities of the laser scanning cytometer (LSC) and adapt this instrument to detect and measure in situ the process of translocation of Bax to mitochondria.

Methods: Human breast carcinoma MCF-7 cells growing on microscope slides were treated with the DNA topoisomerase I inhibitor, camptothecin (CPT). CPT is known to induce apoptosis preferentially of S-phase cells. The cells were fixed and permeabilized on the slides, their DNA was stained with propidium iodide (PI), Bax was detected immunocytochemically with the fluoresceinated antibody, and red and green fluorescence emission was measured by the LSC.

Results: Prior to induction of apoptosis, Bax was uniformly and diffusely dispersed in the cell nucleus and cytoplasm. Its translocation and accumulation in mitochondria in cells undergoing apoptosis were detected and measured by the LSC as the increase in intensity of maximal pixel of Bax immunofluorescence. Bivariate analysis of DNA content versus maximal pixel of Bax fluorescence revealed that the CPT-induced Bax translocation into mitochondria was preferential to S-phase cells. Total cellular Bax immunofluorescence measured by flow cytometry, however, was increased in all phases of the cycle without a preference to S-phase cells.

Conclusion: Changes in abundance and localization of particular proteins that undergo translocation within the cell, leading to their altered local density, may be conveniently detected by the LSC by taking advantage of its morphometric capabilities. Measurement of total cellular Bax immunofluorescence by flow cytometry combined with analysis of its translocation by LSC revealed that apoptosis of S-phase cells induced by CPT was unrelated to overall Bax abundance per cell but correlated with its accumulation in mitochondria.

Background: Flow-sorted plant chromosomes are being increasingly used in plant genome analysis and mapping. Consequently, there is a need for a rapid method for identification of sorted chromosomes and for determination of their purity. We report on optimization of procedures for primed in situ DNA labeling (PRINS) and cycling-PRINS (C-PRINS) for fluorescent labeling of repetitive DNA sequences on sorted plant chromosomes suitable for their identification.

Methods: Chromosomes of barley, wheat, and field bean were sorted onto microscope slides, dried, and subjected to PRINS or C-PRINS with primers for GAA microsatellites (barley and wheat) or FokI repeat (field bean). The following parameters were optimized to achieve the highest specificity and intensity of fluorescent labeling: ratio of labeled versus unlabeled nucleotides, nucleotide concentration, and the number and concentration of primers.

Results: Under optimal conditions, C-PRINS resulted in strong and specific labeling of GAA microsatellites on sorted barley and wheat chromosomes and FokI repeats on sorted field bean chromosomes. The labeling patterns were characteristic for each chromosome and permitted their unequivocal identification as well as determination of purity after sorting, which ranged from 96% to 99%. A standard polymerase chain reaction (PCR) with chromosome-specific primers was not sensitive enough to detect low-frequency contamination.

Conclusions: The results indicate that a single C-PRINS assay with primers that give chromosome-specific labeling pattern is sufficient not only to determine chromosome content of peaks on flow karyotype but also to determine the purity of sorted chromosome fractions. The whole procedure can be performed in less than 3 h on the next day after sorting. Numerous applications are expected in the area of plant flow cytogenetics.