A small-volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four-color flow cytometry

BACKGROUND: Cell permeabilization for the detection of intracellular molecules by flow cytometry is usually incompatible with whole blood. This article describes a new technique for the simultaneous detection of surface antigens and DNA content in rat whole blood. METHODS: In 20 microl of rat whole blood, DNA staining is obtained by permeabilization of cells using a standard red blood cell lysing reagent (Erythrolyse). Immunophenotyping and apoptosis detection by flow cytometry are achieved by using a combination of three surface markers (CD3, CD4, and CD8alpha) and a DNA binding dye (TO-PRO-3). RESULTS: After a 24-h incubation of whole blood with 1 microM dexamethasone, apoptotic lymphocytes were clearly distinguishable from normal lymphocytes by their reduced size and DNA content. The dexamethasone-induced percentage of apoptotic cells was 58.9 +/- 4.6 for CD4+ and 77.4 +/- 2.9 for CD8+ T cells, compared with 12.6 +/- 2.7 for CD4+ and 17.2 +/- 3.5 for CD8+ T cells in the absence of dexamethasone (data from 10 animals with duplicate samples). CONCLUSIONS: We have developed a new technique to permeabilize nucleated cells in microsamples of rat whole blood. The methodology allows simultaneous immunophenotyping and apoptosis detection in rat whole blood.     

A possible new syndrome with growth-hormone secreting pituitary adenoma, colonic polyposis, lipomatosis, lentigines and renal carcinoma in association with familial testicular germ cell malignancy: A case report

Introduction

Cytofluorographic and molecular techniques are effective adjuncts in diagnosing intraocular lymphoma. Primary intraocular lymphoma is an uncommon entity predominantly of B cell origin and rarely with a T cell phenotype. The aim of the present paper is to report a case of a CD8-positive, TCR-α/β-negative intraocular T cell lymphoma and review the literature.

Case presentation

T cell neoplasia was detected based on flow cytometric demonstration of an abnormal T cell population and polymerase chain reactions for immunoglobulin and T-cell receptor rearrangements demonstrating evidence of monoclonality. Flow cytometry revealed a T cell population aberrantly expressing T-cell lineage markers. This T cell population expressed CD2, bright CD3, CD8, bright CD7, CD38, CD69, and variable CD25. T-cell receptor γ gene rearrangement studies demonstrated evidence of T-cell gene rearrangement confirming that the T cells were monoclonal.

Conclusion

We herein report the rare case of a TCR α/β-negative CD8+ intraocular T-cell lymphoma suggestive of gamma/delta origin diagnosed by flow cytometry and polymerase chain reaction.     

Cytokine flow cytometry: multiparametric approach to immune function analysis

More precise quantitation of cellular immune responses has become possible with the advent of single-cell assays of immune function, such as cytokine flow cytometry, enzyme-linked immunospot (ELISPOT), and MHC-peptide multimers. Cytokine flow cytometry is an attractive technique because it allows the detection of responses to whole antigens without regard to MHC restriction, while also collecting additional information on responding cells via multiparameter flow cytometry. In this review, we compare cytokine flow cytometry with other assays of immune function, summarize some of that data that have been collected in various disease states using cytokine flow cytometry, and describe some methodological improvements designed to increase the robustness, throughput, and information content of this technique. We hypothesize that a new generation of automated cytokine flow cytometry assays will allow elucidation of the correlates of protection for diseases involving cellular immunity, through application of these assays in more and large clinical trials.     

FLOW CYTOMETRY AND THE SINGLE CELL IN PHYCOLOGY

Flow cytometers measure light scattering and fluorescence characteristics from individual particles in a fluid stream as they cross one or more light beams at rates of up to thousands of events per second. Flow cytometrically detectable optical signals may arise naturally from algae, reflecting cell size, structure, and endogenous pigmentation, or may be generated by fluorescent stains that report the presence of otherwise undetected cellular constituents. Some flow cytometers can physically sort particles with desired optical characteristics out of the flow stream and collect them for subsequent culture or other analyses. The statistically rigorous, cell‐level perspective provided by flow cytometry has been advantageous in experimental investigations of phycological problems, such as the regulation of cell cycle progression. The capacity of flow cytometry to measure large numbers of cells in large numbers of samples rapidly and quantitatively has been used extensively by biological oceanographers to define the distributions and dynamics of marine picophytoplankton. Recent work has shown that flow cytometry can be used to elucidate relationships between the optical properties of individual cells and the bulk optical properties of the water they live in, and thereby may provide an explicit link between algal physiology and global biogeochemistry. Unfortunately, commercially available flow cytometers that are optimized for biomedical applications have a limited capacity to analyze larger phytoplankton. To circumvent these limitations, many investigators are developing flow cytometers specifically designed for analyzing the broad range of sizes, shapes, and pigments found among algae. These new instruments can perform some novel measurements, including simple fluorescence excitation spectra, detailed angular scattering measurements, and in‐flow digital imaging. The growing accessibility and power of flow cytometers may allow the technology to be applied to a wider array of problems in phycology, including investigations of nonplanktonic and multicellular algae, but also presents new challenges for effectively analyzing the large quantity of multiparameter data produced. Ultimately, the detection of molecular probes by flow cytometry may allow single‐cell taxonomic and physiological information to be garnered for a variety of algae, both in culture and in nature.     

Polychromatic flow cytometry: a rapid method for the reduction and analysis of complex multiparameter data.

BACKGROUND: Recent advances in flow cytometry have resulted in the development of reliable techniques for performing polychromatic (5-17 color) flow cytometry analysis. However, the data reduction and analysis involved in the resolution of hundreds of possible cellular subphenotypes identified, using a single polychromatic flow cytometry staining panel, presents a major obstacle to the successful application of this technology. METHODS: To generate two distinct collections of T cell populations with differentially expressed surface markers, cryopreserved lymph node cells from 5 melanoma patients vaccinated with the modified gp100(209-2M) melanoma peptide were stimulated with cognate peptide and cultured in either IL-21 + low-dose IL-2 or IL-15 + low-dose IL-2. In vitro stimulated (IVS) cells were interrogated using 8-color flow cytometry. Data were analyzed using Winlist Hyperlog and FCOM software, and 32 T cell subsets were resolved for each culture condition. Hierarchical clustering analysis was applied to the relative percentages of each subphenotype for both IVS conditions to determine if unique cell surface marker expression signatures were produced for each IVS culture. RESULTS: Sequential data analysis using Hyperlog and FCOM demonstrated that lymphocytes cultured in IL-21 + IL-2 had a distinctively different set of subphenotype signatures compared to cells grown in IL-15 + IL-2 for all 5 patients. Importantly, subsequent cluster analysis of all 32 subphenotype frequencies in each IVS test condition for all 5 patients reproducibly demonstrated that cellular subphenotypes produced after IL-21 + IL-2 IVS partitioned separately from subphenotypes produced by IL-15 + IL-2 IVS. CONCLUSIONS: The integrated sequential use of Hyperlog and FCOM software with cluster analysis algorithms for the reduction and analysis of polychromatic flow cytometry data produces an effective, rapid technique for the assessment of complex patterns of subphenotype expression between and within multiple test samples. This approach to data analysis may enhance the use of polychromatic flow cytometry for both research and clinical applications.     

Sheath fluid control to permit stable flow in rapid mix flow cytometry.

BACKGROUND: Flow cytometry is a potentially powerful tool to analyze the kinetics of ligand binding, cell response and molecular assembly. The difficulty in adding reactant to cells, achieving adequate mixing, delivering those cells to the laser focal point and establishing stable flow, has historically limited flow cytometry to systems with reactions times longer than 5 s. With the advent of automated syringes and flow injection methods, sample injection times shorter than 1 s have become routine. However, an inherent problem in acquiring time courses starting under 1 s is that rapid sample introduction through the flow tip to the detection point perturbs laminar flow. The purpose of this work was to determine if stable flow could be reestablished more quickly if the sheath flow was reduced during sample introduction, returning to normal sheath and sample rates afterward. METHODS: We used programmable syringes and valves to control sample mixing as well as sheath and sample delivery through the flow tip to the detection point for stream-in-air detection. Stable flow was monitored by mean particle fluorescence during sample introduction. RESULTS: With no sheath reduction, stable flow recovered after more than 1 s. By reducing sheath flow during the short period (300 msec) of sample mixing and delivery, stable laminar flow recovered within 200 msec. CONCLUSIONS: This use of automated syringes to control both sheath and sample flow provides a potential for robust sample handling applicable to kinetic as well as high throughput flow cytometric analysis.     

Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy

BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.     

Multimodality imaging of endothelial progenitor cells with a novel multifunctional probe featuring positive magnetic resonance contrast and near-infrared fluorescence

The multimodal strategy incorporating T1-weighted magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence imaging can complement their strengths to provide images with high sensitivity and spatial resolution for noninvasively and dynamically monitoring endothelial progenitor cells (EPCs) in potential EPC-dominated therapies. Here we report the development of a protein-based imaging probe, bCD-PLL-Cy5.5 Conjugate 1, in which the bacterial cytosine deaminase (bCD) protein was modified with poly-l-lysine (PLL) that is labeled with imaging reporters, including T1-weighted MRI contrast chelator and NIR fluorophore. Conjugate 1 showed low cytotoxicity in EPCs isolated from the rabbit peripheral blood. The normalized cell viability was maintained above 90% after incubation for 1 to 5 days. Fluorescence microscopy of live cells indicated rapid cellular uptake of Conjugate 1 into EPCs in 15 minutes, and flow cytometry studies demonstrated the time-dependent internalization of Conjugate 1 with maximum uptake 48 hours after the treatment. MRI of phantoms demonstrated significant reduction of the T1 value of the EPC pellet that was pretreated with 2 μM of Conjugate 1 for 24 hours. Our preliminary data suggest that as a multimodal imaging contrast medium, Conjugate 1 offers a promising imaging probe for tracking the delivery and therapeutic response of EPCs in vivo.     

Detection, mapping, and quantification of single walled carbon nanotubes in histological specimens with photoacoustic microscopy

Aims

In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds).

Materials and Methods

Optical-resolution (OR) and acoustic-resolution (AR) - Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy).

Results

Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections.

Conclusions

The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs.     

Photosynthetic electron transport: Emergence of a concept, 1949–59

Historically, two main concepts guided research into possible mechanisms of light-induced atomic rearrangements in oxygenic photosynthesis: Photodecomposition of CO₂ and photodecomposition of water. Both concepts envisioned photoinduced transfers of cumbersome whole atoms and not, as is currently held, photoinduced electron transfers. Early proposals for light-induced electron transfers were relegated to obscurity because they were speculative ideas, not supported by meaningful experimental findings and tied to hypothetical and ephemeral schemes. The concept of photoinduced rearrangements of whole atoms rather than electrons was so well entrenched that it was even invoked to explain their findings by the discoverers of the Hill reaction and cyclic photophosphorylation. The light-induced electron flow concept gained acceptance in photosynthesis research only with the discovery of non-cyclic photophosphorylation in which ATP formation is coupled with electron transport to ferredoxin/NADP⁺ or to artificial substitutes like ferricyanide.     

Recent progress in mass spectrometry for single-cell metabolomics

Metabolites are the end products of cellular vital activities and can reflect the state of cellular to a certain extent. Rapid change of metabolites and the low abundance of signature metabolites cause difficulties in single-cell detection, which is a great challenge in single-cell metabolomics analysis. Mass spectrometry (MS) is a powerful tool that uniquely suited to detect intracellular small-molecule metabolites and has shown good application in single-cell metabolite analysis. In this mini-review, we describe three types of emerging technologies for MS-based single-cell metabolic analysis in recent years, including nano-ESI-MS based single-cell metabolomics analysis, high-throughput analysis via flow cytometry, and cellular metabolic imaging analysis. These techniques provide a large amount of single-cell metabolic data, allowing the potential of MS in single-cell metabolic analysis is gradually being explored and is of great importance in disease and life science research.     

Phytoplankton monitoring by high performance flow cytometry: a successful approach?

BACKGROUND: Regular phytoplankton monitoring in Dutch coastal waters is performed as an indicator of the ecological state of these waters. The monitoring program is focused on temporal and spatial changes of species composition and abundance. Flow cytometry has been introduced to provide additional information, to improve ecosystem understanding, and to increase the efficiency of analysis and reportage. METHODS: Phytoplankton community abundance and composition were routinely determined by flow cytometry and microscopy at six locations in the North Sea over three annual cycles between 2000 and 2003. Supplementary measurements were also made for fluorescence (chlorophyll-a and other pigments) and, in combination with flow cytometric and microscopic data, were used to determine phytoplankton abundance and composition as a function of their size distribution. Real-time imaging of species was also used to identify species on the basis of their flow cytometric optical characteristics. RESULTS: Flow cytometric analysis took 15 min on average. Analysis including data processing, and Web site reportage took less than 1 h. Phytoplankton concentrations (cells/ml), biomass (fluorescence/ml), and concentration of phycoerythrin- or phycocyanin-containing cells (cells/ml) as a function of their algal size were produced every 2 weeks on average. The phytoplankton integrated annual concentration and biomass were used as ecological indicators for overall phytoplankton status. Real-time imaging of cells in flow enabled the identification of dominant species and was applied as an early warning system for Phaeocystis spp. CONCLUSIONS: The reproducibility and count precision due to the large number of observations of the flow cytometric technique provided reliable data for monitoring long-term trends. Flow cytometrically based analyses extended the lower detection limit (<0.5 microm) of analysis beyond the capabilities of other techniques such as the relation between small and larger phytoplankton, the relation between cell counts and biomass as a function of cell size, but also the ability to monitor and report on blooms of harmful algae. A good correlation was found between concentrations (cells/ml) measured by flow cytometry and microscopy. In practice, flow cytometric analysis of a single marine sample took 15 min on average.     

用YO-PRO-1和PI联合染色定量检测细胞凋亡

经不同浓度staurosporine处理诱导凋亡的G7细胞样品,分别用YO-PRO-1/PI和AV/PI进行荧光染色,借助流式细胞仪检测凋亡情况,将两种检测方法得到的结果进行统计学分析显示,二者有显著的相关性(r=0.9659,P<0.01),且没有显著性差异(P<0.05);另外,上述凋亡细胞样品经YO-PRO-1/PI染色后在荧光显微镜下计数凋亡细胞比例的结果与AV/PI流式细胞仪的检测结果也有显著的相关性(r=0.9903,P<0.01),且没有显著性差异(P<0.05)。以上这些结果表明,用YO-PRO-1/PI对细胞进行染色、借助流式细胞仪和荧光显微镜均能准确地检测细胞凋亡,可替代AV/PI流式细胞仪方法用于细胞凋亡的检测。     

Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations

Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples.     

Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed (1-2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial-temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type.     

Combination of automated high throughput platforms, flow cytometry, and hierarchical clustering to detect cell state.

BACKGROUND: This study examined whether hierarchical clustering could be used to detect cell states induced by treatment combinations that were generated through automation and high-throughput (HT) technology. Data-mining techniques were used to analyze the large experimental data sets to determine whether nonlinear, non-obvious responses could be extracted from the data. METHODS: Unary, binary, and ternary combinations of pharmacological factors (examples of stimuli) were used to induce differentiation of HL-60 cells using a HT automated approach. Cell profiles were analyzed by incorporating hierarchical clustering methods on data collected by flow cytometry. Data-mining techniques were used to explore the combinatorial space for nonlinear, unexpected events. Additional small-scale, follow-up experiments were performed on cellular profiles of interest. RESULTS: Multiple, distinct cellular profiles were detected using hierarchical clustering of expressed cell-surface antigens. Data-mining of this large, complex data set retrieved cases of both factor dominance and cooperativity, as well as atypical cellular profiles. Follow-up experiments found that treatment combinations producing "atypical cell types" made those cells more susceptible to apoptosis. CONCLUSIONS Hierarchical clustering and other data-mining techniques were applied to analyze large data sets from HT flow cytometry. From each sample, the data set was filtered and used to define discrete, usable states that were then related back to their original formulations. Analysis of resultant cell populations induced by a multitude of treatments identified unexpected phenotypes and nonlinear response profiles.     

Influence of EDTA and heparin on lipopolysaccharide binding and cell activation, evaluated at single-cell level in whole blood

BACKGROUND: The use of whole blood (WB) in studying lipopolysaccharide (LPS)-induced cellular activation preserves the milieu in which LPS-cell interaction occurs in vivo. However, little information is available on using such a system at a single-cell level. We evaluated LPS binding and cell activation in WB by using flow cytometry. The influence of heparin or EDTA as anticoagulants was also addressed. METHODS: Blood was obtained from healthy donors in EDTA and/or heparin tubes. Biotinylated LPS (LPSb) was used to evaluate cell binding of LPS in WB. Cells were surface stained with appropriate antibodies and LPSb was detected by adding streptavidin-allophycocyanin (APC). LPS-induced activation was evaluated by the expression of surface activation markers and by the detection of intracellular tumor necrosis factor-alpha (TNF-alpha). RESULTS: LPSb bound promptly to monocytes in EDTA- and heparin-treated blood. In EDTA-treated blood, membrane-bound LPSb decreased after 60 min of incubation, whereas it remained detectable in heparinized blood during the 6 h of incubation. LPS induced TNF-alpha and enhanced the expression of HLA-DR in monocytes, as well as the expression of CD69 in T and B lymphocytes. Induction of both TNF-alpha in monocytes and CD69 in lymphocytes was more efficient in heparinized blood. CONCLUSION: Detection of membrane-bound LPSb on monocytes differed in EDTA or heparin-treated blood, and cell activation was better obtained in heparinized blood.     

Flow cytometry, a very useful technique for the characterization of intestinal membrane vesicles

The plasma membrane of enterocytes comprises two structurally and functionally distinct domains. These are the apical brush border, containing digestive hydrolases and glycocalyx, and the basolateral domain, characterized by other specific markers. Using a fast and easy subcellular fractionation, we purified four membrane vesicle fractions from rabbit small intestinal mucosa: brush border, basolateral, rough endoplasmic reticulum and Golgi + smooth endoplasmic reticulum. Using flow cytometry, the fluorescence polarization of diphenylhexatriene was determined in brush border and in basolateral + Golgi + smooth endoplasmic reticulum membrane fractions in order to investigate changes in the membrane fluidity of both fractions and to compare the results obtained with those of spectroscopic techniques. Moreover, it was possible with flow cytometry to detect and quantify basolateral and brush border markers by using polyclonal and monoclonal antibodies. The advantages of flow cytometry in the detection of brush border membrane markers found in small amounts in the basolateral domain are discussed. Finally, flow cytometry holds great promise for the analysis and sorting of subcellular fractions.