Flow cytometry of isolated mitochondria during development and under some pathological conditions

Mitochondria play an essential role in the generation of the energy needed for eukaryotic cell life and in the release of molecules involved in initiation of cell death. Here we review the changes in isolated mitochondrial fluorescent populations as distinguished by flow cytometry during postnatal development and their regulation by thyroid hormones and catecholamines. The use of flow cytometry in the study of mitochondrial changes occurring under hypothyroidism, alcohol abuse and aging is also reviewed.     

Fundamentals of flow cytometry

Flow cytomelers arc instruments that arc used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytomcters are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/ particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytomety is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.     

Real-time on-line flow cytometry for bioprocess monitoring

As the understanding of variation is the key to a good process and product quality one should pay attention to dynamics on the single-cell level. The basic idea of this approach was to qualify and quantify variations on the single-cell level during bioreactor cultivations by monitoring the expression of an eGFP tagged target protein (human membrane protein) using fully automated real-time, flow injection flow cytometry (FI-FCM). The FI-FCM system consists of a sampling- and defoaming- as well as of a dilution-section. It allows a very short monitoring interval (5 min) and is able to dilute the reactor sample by a factor ranging up to more than 10,000.In bioreactor cultivations of recombinant Pichia pastoris expressing the eGFP tagged target protein, high correlations (R² ≥ 0.97) between the FI-FCM fluorescent signal and other, however, population-averaged fluorescence signals (off-line fluorescence, in situ fluorescence probe) were obtained. FI-FCM is the only method able to distinguish between few cells with high fluorescence and many cells with low fluorescence intensity and proved that cells differ significantly from each other within the population during bioreactor cultivations. Single-cell fluorescence was distributed over a broad range within the cell population. These distributions strongly suggest that (a) the AOX-I promoter is leaky and (b) a fraction of the population is able to express more protein of interest within shorter time and (c) a fraction of the population does not express the fusion protein at all. These findings can help in the selection of high producing, stable strains. To show the platform-independency of the system, it has successfully been tested during bioreactor cultivations of three different strains (P. pastoris, Saccharomyces cerevisiae, Escherichia coli).Along with its applications in PAT, the FI-FCM could be used as a platform-independent (prokaryotes and eukaryotes) method in various other applications; for example in the closed-loop-control of bioprocesses using different kinds of fluorescent reporters, (waste- and drinking-) water analysis, clone selection in combination with FACS or even for surgery applications.     

Isolation of functional pure mitochondria by superparamagnetic microbeads

Isolation of mitochondria by current methods relies mainly on their physicochemical properties. Here we describe an alternative approach to obtain functional mitochondria from human cells in a fast, reproducible, and standardized procedure. The new approach is based on superparamagnetic microbeads conjugated to anti-TOM22 antibody. The bead conjugates label the cytoplasmic part of the human mitochondrial membrane protein TOM22 and, thus, allow for a gentle isolation of mitochondria in a high gradient magnetic field. By comparing the MACS (magnetic cell separation) approach with mitochondria isolation methods using differential centrifugation and ultracentrifugation we demonstrate that the MACS approach provides the highest yield of isolated mitochondria. The quality, enrichment, and purity of mitochondria isolated with this protocol are comparable to mitochondria obtained using the ultracentrifuge method, and a typical separation procedure takes only approximately 1 to 2 h from initial cell homogenization. Mitochondria isolated with the new approach are sufficient for protein import, blue native gel electrophoresis, and other mitochondrial assays.     

流式细胞术计数细菌的研究

目的探讨用流式细胞术进行细菌计数。方法检测被链球菌α-34抑止而死亡的化脓性链球菌32309-2数量。结果实验管MFI值明显比对照管强,实验管本身的MFI强度随着时间的增加而增强,而对照管则基本上变化不是很大;到24h后,对照管MFI已达1．01,而对照管还是维持在0．4左右,两者T检验P〈0．01。结论用PI染色,可用流式细胞术检测死亡细菌。结果可靠,用时短,极大地简化了实验步骤。     

Cytokeratin analysis of breast and leukemia tumor cell lines by flow cytometry

Using four human tumor cell lines, MCF-7 and T47-D from breast tumors, MOLT-4 and K-562 from leukemia, flow cytometric DNA analysis of pure and mixed cell population was performed using monoclonal antibodies to cytokeratin to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratins. Surprisingly, on pure or mixed K-562 cells, we found positive labeling with KL1, CK8, and CK18 antibodies (results confirmed by immunocytology). This preliminary study has allowed a DNA analysis on epithelial cells of human breast tumors.     

Systematic misestimation of cell subpopulations by flow cytometry: A mathematical analysis

Various sources of variability in flow cytometric determination of cell concentration have previously been investigated with respect to andrologic applications. Although common aspects related to the variation between samples, variation between operators, and accuracy have been extensively studied, specific sources of false-count estimation have found less attention. In particular, a major and well-recognized source of misestimation of cell counts (i.e., contamination of the sample by non-sperm particles) has not to date been characterized in detail. We show here by means of original mathematical research that not only the cell counts but also the percentages of cells expressing different fluorescence patterns are affected by the presence of alien particles often neglected in studies involving flow cytometric characterization. We demonstrate that there is a systematic overestimation in the proportion of unstained (viable) cells detected by flow cytometry in cases where the non-sperm particles are not excluded from analysis by additional identification other than light-scatter characteristics. Moreover, we provide an exact mathematical estimate for the magnitude of this overestimation, and we discuss the consequences for diagnostic applications and studies on sperm physiology, specifically for studies on sperm capacitation and evaluation of cryopreserved semen. Finally, equations are derived for the correction of the flow cytometric values for use in practical applications.     

Size measurements on isolated rat heart cells using coulter analysis and light scatter flow cytometry

Isolated ventricular muscle cells from the adult rat heart have been examined by both Coulter analysis and light scatter flow cytometry. The dispersed cell preparations contain two main cell types: viable, rod-shaped cells and damaged, round cells. Coulter analytical techniques provided statistical data on cell volume for both cell types. The contribution of each population to the Coulter pulse height distributions were separated by a subtraction method using data obtained from digitonin-treated preparations that contain only round cells. A shape factor for cells aligned with the flow direction was computed from light microscope measurements and the effects of cell orientation within the Coulter aperture were approximately assessed. The estimated volumes for intact myocytes compare favourably with those reported in the literature. No significant size difference was observed between fresh and fixed cells.Narrow angle, forward light scatter measurements were made on individual cells flowing across a focused laser beam. Both scatter pulse height and pulse width (pulse duration) distributions were collected. Values for myocyte length calculated from pulse width information agree well with published data and confirm that the hydrodynamic forces in the flow system produced alignment of the cells with the flow direction. Scatter pulse width distributions reveal two distinct peaks assignable to either rod or round cells. Preliminary electronic gating experiments, using pulse height signals, suggest that signals derived from round cells could be eliminated entirely using a gating regime based on pulse width. This would enable flow cytometric measurements to be made on only the intact myocytes present in heterogeneous preparations.     

Mucin-lectin interactions assessed by flow cytometry

The O-glycosylated domains of mucins and mucin-type glycoproteins contain 50-80% of carbohydrate and possess expanded conformations. Herein, we describe a flow cytometry (FCM) method for determining the carbohydrate-binding specificities of lectins to mucin. Biotinylated mucin was immobilized on streptavidin-coated beads, and the binding specificities of the major mucin sugar chains, as determined by GC-MS and MALDI-ToF, were monitored using fluorescein-labeled lectins. The specificities of lectins toward specific biotinylated glycans were determined as controls. The advantage of flexibility, multiparametric data acquisition, speed, sensitivity, and high-throughput capability makes flow cytometry a valuable tool to study diverse interactions between glycans and proteins.     

Quantification of nuclear DNA and G-C content in marine macroalgae by flow cytometry of isolated nuclei

Summary The amounts of nuclear DNA in ten species of seaweeds belonging to the Rhodophyceae, Phaeophyceae, and Chlorophyceae were determined by flow cytometric analysis of nuclei isolated from protoplasts. Genome size was determined from the fluorescence of the nuclei stained with ethidium bromide. The size of the nuclear genome ranged from 0.13 pg per cell in the 1 C population ofUlva rigida to 3.40 pg per cell in the 2 C population ofSphacelaria sp. GC% analysis was based on staining with either Hoechst 33342 or mithramycin A, two fluorochromes specific for the bases A-T and G-C, respectively. Two models were used for the estimation of the proportion of guanine plus cytosine in the nuclear genome. The first one was based on the linear relationships mithramycin A fluorescence/G-C content and ethidium bromide fluorescence/total DNA content. The second model, based on the curvilinear relationships Hoechst 33342 fluorescence/A-T content and mithramycin A fluorescence/G-C content, resulted in comparatively more homogenous and consistent data and appears more accurate. Comparison with previous reports from other methods for the physical investigation of nuclear genomes shows that flow cytometry of nuclei isolated from protoplasts is an accurate, convenient and robust technique to assay for genome sizes and base pair composition in marine macroalgae.Abbreviations A-T nucleic bases adenine and thymine - CRBC chicken red blood cell - FALS forward-angle light scatter - G-C nucleic bases guanine and cytosine - SEIM sorbitol enzymatic incubation medium - SWIM sea water incubation medium - Tm thermal denaturation temperature of DNA     

A flow cytometry approach for quantitative analysis of cellular phosphatidylserine distribution and shedding

Phospholipids are asymmetrically distributed across the membrane of all cells, including red blood cells (RBCs). Phosphatidylserine (PS) is mainly localized in the cytoplasmic membrane leaflet, but during RBC ageing it flip-flops to the external leaflet—a process that is increased in certain pathological conditions (e.g., β-thalassemia). PS externalization in RBCs mediates their phagocytosis by macrophages and removal from the circulation. PS is usually measured by flow cytometry and is reported as the percentage of cells with external PS. In the current study, we developed a novel two-step flow cytometry procedure to quantitatively measure not only the external PS but also the intracellular and shed PS. In this method, PS is first bound to fluorescent annexin V, and then the residual nonbound annexin is quantified by binding to PS exposed on apoptotic cells. Using this method, we measured 1.1 ± 0.2 and 0.12 ± 0.04 μmol inner and external PS, respectively, per 10⁷ normal RBCs. Thalassemic RBCs demonstrated increased PS externalization (1.7-fold) and shedding (11-fold) that was accompanied by lower intracellular PS (31%). These results suggest that quantitative flow cytometry of PS could have a diagnostic value in evaluating the pathology of RBCs in hemolytic anemias associated with increased PS externalization and shortening of the RBC life span.     

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.     

Nicorandil protects cardiac mitochondria against permeability transition induced by ischemia-reperfusion

Ischemia followed by reperfusion is known to negatively affect mitochondrial function by inducing a deleterious condition termed mitochondrial permeability transition. Mitochondrial permeability transition is triggered by oxidative stress, which occurs in mitochondria during ischemia-reperfusion as a result of lower antioxidant defenses and increased oxidant production. Permeability transition causes mitochondrial dysfunction and can ultimately lead to cell death. A drug able to minimize mitochondrial damage induced by ischemia-reperfusion may prove to be clinically effective. We aimed to analyze the effects of nicorandil, an ATP-sensitive potassium channel agonist and vasodilator, on mitochondrial function of rat hearts and cardiac HL-1 cells submitted to ischemia-reperfusion. Nicorandil decreased mitochondrial swelling and calcium uptake. It also decreased reactive oxygen species formation and thiobarbituric acid reactive substances levels, a lipid peroxidation biomarker. We thus confirm previous reports that nicorandil inhibits mitochondrial permeability transition and demonstrate that nicorandil inhibits this process by preventing oxidative damage and mitochondrial calcium overload induced by ischemia-reperfusion, resulting in improved cardiomyocyte viability. These results may explain the good clinical results obtained when using nicorandil in the treatment of ischemic heart disease.     

New method to characterize microbial diversity using flow cytometry

The majority of microorganisms have yet to be cultivated and represent a vast uncharacterized and untapped resource. Here, we report the utilization of a combination of flow cytometry, cultivation, and molecular genetics to develop new methodologies to access and characterize biodiversity in microbial samples. We demonstrate that fluorescent dyes and combinations of dyes can selectively stain portions of bacterial populations that can be isolated as sub-populations using fluorescence-activated cell sorting (FACS). Microbial sub-populations obtained by FACS differ substantially from the original microbial population, as demonstrated by denaturing gradient gel electrophoresis and determination of 16S rRNA gene sequences. These sub-populations can subsequently be used to inoculate microbial growth media, allowing the isolation of different microbial species from those that can be readily cultivated from the original sample using the same microbial growth media. When this technique was applied to the analysis of activated-sludge and Yellowstone Lake hydrothermal vent samples, comparative analysis of 16S rDNA sequences revealed that FACS allowed the detection of numerous bacterial species, including previously unknown species, not readily detectable in the original sample due to low relative abundance. This approach may result in a convenient methodology to more thoroughly characterize microbial biodiversity.     

Evaluation of fresh and frozen-thawed fowl semen by flow cytometry

The aim of this study was to assess the spermatozoal viability, acrosome integrity, mitochondrial activity, and DNA status in the frozen-thawed fowl semen with the use of flow cytometry. The experiment was carried out on 10 sexually adult roosters of meat type line Flex. The semen was collected three times a week by dorso-abdominal massage method, then pooled and subjected to cryopreservation using “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. For cytometric analysis the fresh and frozen-thawed semen was extended with EK diluent to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with dual fluorescent probes SYBR-14 and propidium iodide (PI). Acrosomal damages were evaluated using phycoerythrin-conjugated lectin PNA from Arachis hypogaea. The percentage of live spermatozoa with functional mitochondria was estimated using Rhodamine 123 (R123) and PI. The spermatozoal DNA integrity was measured by sperm chromatin structure assay (SCSA). The freezing-thawing process decreased the viability, mitochondrial activity in the chicken sperm and increased the percentage of dead cells with ruptured and intact acrosomes, and also the percentage of spermatozoa with fragmented DNA. In conclusion, the present study indicates that fluorescent staining and flow cytometry may be useful for assessment of the changes of fowl semen quality caused by cryopreservation process. This technique allows precise examination of spermatozoa functional characteristic in a very short time.     

Toxinological studies of the venom from Cassiopea xamachana nematocysts isolated by flow cytometry

The tentacle epithelial tissue of Cassiopea xamachana contains nematocysts and symbiotic algal particles. These two structures were dissociated, analyzed and sorted by flow cytometry. A simple separating method was developed utilizing the algal chlorophyll autofluorescence and the nematocysts' fluorescence after the uptake of fluorescent stains. A five-fold increase in mouse lethality; significantly more potent hemolytic and cytosensing activities; as well as a cleanup in the capillary electropherogram and SDS gel profiles for the crude nematocyst venom preparations prepared by fluorescence activated cell sorting (FACS), was observed relative to alternative methods. Because the hemolytic potency of pre-sorting nematocyst venom was minimal and the post-sorting counterpart was significantly positive, the possibility that algae inhibited the venom's toxinological activity was considered.     

Identification of two human sperm populations using flow and image cytometry

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder (“marginal population”). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation. We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion. We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide. © 1994 Wiley-Liss, Inc.     

Advances in flow cytometry for sperm sexing

This review presents the key technological developments that have been implemented in the 20 years since the first reports of successful measurement, sorting, insemination and live births using flow cytometry as a proven physical sperm separation technique. Since the first reports of sexed sperm, flow technology efforts have been largely focused on improving sample throughput by increasing the rate at which sperm are introduced to the sorter, and on improving measurement resolution, which has increased the proportion of cells that can be reliably measured and sorted. Today, routine high-purity sorting of X- or Y-chromosome-bearing sperm can be achieved at rates up to 8000 s⁻¹ for an input rate of 40,000 X- and Y- sperm s⁻¹. With current protocols, straws of sex-sorted sperm intended for use in artificial insemination contain approximately 2 × 10⁶ sperm. The sort rate of 8000 sperm s⁻¹ mentioned above corresponds to a production capacity of approximately 14 straws of each sex per hour per instrument.     

Phenylarsine oxide induces the cyclosporin A-sensitive membrane permeability transition in rat liver mitochondria

This paper reports an investigation on the effects of the hydrophobic, bifunctional SH group reagent phenylarsine oxide (PhAsO) on mitochondrial membrane permeability. We show that PhAsO is a potent inducer of the mitochondrial permeability transition in a process which is sensitive to both the oxygen radical scavanger BHT and to cyclosporin A. The PhAsO-induced permeability transition is stimulated by Ca²⁺ but takes place also in the presence of EGTA in a process that maintains its sensitivity to BHT and cyclosporin A. Our findings suggest that, at variance from other known inducers of the permeability transition, PhAsO reacts directly with functional SH groups that are inaccessible to hydrophilic reagents in the absence of Ca²⁺.