A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry

It has been demonstrated that ELF97-phosphate (ELF-P) is a useful tool to detect and quantify phosphatase activity of phytoplankton populations at a single cell level. Recently, it has been successfully applied to marine heterotrophic bacteria in culture samples, the cells exhibiting phosphatase activity being detected using epifluorescence microscopy. Here, we describe a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF-P hydrolysis, allowing the detection of phosphatase positive bacteria, using flow cytometry. Bacteria from natural samples must be disaggregated and, in oligotrophic waters, concentrated before they can be analyzed by flow cytometry. The best efficiency for disaggregating/separating bacterial cell clumps was obtained by incubating the sample for 30 min with Tween 80 (10 mg l(-1), final concentration). A centrifugation step (20,000 g; 30 min) was required in order to recover all the cells in the pellet (only 7+/-2% of the cells were recovered from the supernatant). The cells and the ELFA precipitates were resistant to these treatments. ELFA-labelled samples were stored in liquid nitrogen for up to four months before counting without any significant loss in total or ELFA-labelled bacterial cell abundance or in the ELFA fluorescence intensity. We describe a new flow cytometry protocol for detecting and discriminating the signals from both ELFA and different counterstains (4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) necessary to distinguish between ELFA-labelled and non ELFA-labelled heterotrophic bacteria. The method has been successfully applied in both freshwater and marine samples. This method promises to improve our understanding of the physiological response of heterotrophic bacteria to P limitation.     

A fast cell sampler for flow cytometry

A simple device has been developed for delivering samples into a flow cytometer. Designed with economy, simplicity, and flexibility in mind, this device, having only one moving part, can be used for sample volumes as small as 20 microliter, for virtually any form of cell sample container, and for a wide range of cell concentrations. It consists essentially of a lever-operated disc valve that allows the cell sample to be loaded into a loop of tubing and then to be injected into the cytometer nozzle under pressure from a saline source. The sampler has lifted the maximum analytical throughput of a FACS II cell sorter to better than 120 samples per hour.     

Modified histogram subtraction technique for analysis of flow cytometry data

Analysis of flow cytometry histogram data by the subjective selection of an integration window can be a tedious and time-consuming task and is often inaccurate. A new method for automated calculation of the percent positive from immunofluorescence histograms is presented. This new method is a modification of the currently used method of channel-by-channel histogram subtraction. Its accuracy is compared to that of the channel-by-channel histogram subtraction method and to another currently used automated method, which selects an integration window by finding the channels that contain the most fluorescent 2% of a control histogram. The new histogram subtraction method is objective, easy to use, and is more accurate than other currently used automated analysis methods. PASCAL source code is given for each method of analysis.     

A rapid method for evaluation of cell number and viability by flow cytometry

A simple, rapid and reliable method has been developed for assessing the number and viability of cells, as well as cell size, in suspension culture by the use of flow cytometry. Propidium iodide exclusion is used for viability determination and fluorescent beads serve as an internal standard for cell enumeration. The main advantages of this method are its ability to handle a large number of samples with a high degree of precision and its specificity in detecting viable cells quantitatively in a heterogeneous culture of living and dead cells and debris. The method shows only a fraction of the variation found in the haemacytometer/trypan blue counting method due to its very low operator dependence. CHO - Chinese hamster ovary; FCS - Foetal calf serum; FS - Forward scatter light; MTT - 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; NCS - newborn calf serum; PBS - Phosphate buffered saline; PI - Propidium iodide; SS - Side scatter light. This revised version was published online in July 2006 with corrections to the Cover Date.     

A protocol to effectively create single cell suspensions of adherent cells for multiparameter high-throughput flow cytometry

High-throughput flow cytometry of adherent cells is difficult because the creation of single cell suspensions can damage cells and yield artificial results. We describe a protocol to increase the single cell suspension yield of adherent human cells without injury. Doxorubicin, a cytotoxic agent, was administered to adherent human pancreatic carcinoma cell lines (Panc-1 and AsPC-1) to produce alterations in the cell cycle and intracellular protein expression. The cells in 96-well plates were disassociated using a collagenase and trypsin mixture. Fluorescence-activated high-throughput flow cytometry evaluated cellular viability as well as surface and intracellular protein expression. Cell cycle analysis was performed using 7-aminoactinomycin D and intracellular protein characterization was performed using a fluorescein-labeled monoclonal antibody against activated caspase-3. The collagenase–trypsin-based protocol increased single cell events from 31.9 ± 0.5% using trypsin alone (standard) to a range of 62.1% to 85.5% without adversely affecting viability. High-throughput flow cytometry demonstrated that the addition of collagenase to the disassociation solution not only permitted significantly higher rates of single cell creation, but it did not negatively affect the doxorubicin-induced protein expression. This protocol allows for expedient and effective disassociation of adherent human cells in order to investigate alterations in specific cellular enzymes and pathways.     

Discrimination of eukaryotic phytoplankton cell types from light scatter and autofluorescence properties measured by flow cytometry

Flow cytometric methods for recognizing several groups of eukaryotic marine phytoplankton were tested using 26 laboratory cultures. Each culture was divided into three aliquots, and these samples were analyzed for 1) Coulter volume; 2) light scatter (magnitude and polarization properties of forward scattered light and magnitude of right-angle scattered light) and autofluorescence emission (phycoerythrin and chlorophyll); and 3) autofluorescence excitation (by 488 nm and 515 nm light). Three kinds of cells could be easily distinguished from others in the culture collection: 1) The two cryptophytes and the rhodophyte had high phycoerythrin/chlorophyll ratios; 2) the two coccolithophores depolarized forward scattered light; and 3) the two pennate diatoms scattered only a relatively small amount of light in the forward direction compared with that at right angles. Mean chlorophyll fluorescence excited by blue light relative to that excited by green light was highest in the four chlorophytes, but there was overlap between some of these and some other kinds of cells. Unresolved cell types included centric diatoms, dinoflagellates, and naked coccolithophores. Forward light scatter and Coulter volume were closely related (except for the pennate diatoms) over a range of about 0.01 to 30 pL (equivalent spherical diameter about 3 to 40 microns), according to a logarithmic function.     

A new method for fast blood cell counting and partial differentiation by flow cytometry

A new blood counting method by flow cytometry is described which determines absolute counts and relative proportions of erythrocytes, reticulocytes, thrombocytes, lymphocytes and granulocytes from one sample of saline diluted human or animal blood. Staining time is 2 to 5 min and measuring time between 1 and 2 additional minutes. Measured simultaneously are the electrical cell volume, the green and optionally also the red fluorescence of the transmembrane potential sensitive dye 3,3-dihexyloxacarbocyanine DiOC6(3) and the RNA/DNA stain acridine orange (AO). Work is under way to fully automate staining, measurement and data evaluation. The use of stains by which blood cell counting and biochemical analysis can be combined offers new possibilities for routine blood cell counting without requirement for additional time. The potential of such stains is that pathologic cell conditions which are not, or not yet reflected in the cell count may be earlier detectable by biochemical stains.     

Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry.

Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non-vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7-amino-actinomycin D (7-AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7-AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7-AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)-fluorescence dual-color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7-AAD was used on fluoresceinisothiocyanate (FITC) and PE surface-labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.     

Multicolour flow cytometry analyses and autofluorescence in chlorophytes: lessons from programmed cell death studies in Chlamydomonas reinhardtii

Flow cytometry is a valuable tool in phycological studies. However, endogenous cellular compounds like nicotinamide adenine dinucleotide and chlorophyll a and b autofluoresce, potentially interfering with fluorescent markers. Furthermore, autofluorescent properties are not uniform across algae, nor are their effects consistent in different cytometers. The choice of instrument and fluorescent marker, therefore, requires careful consideration. We investigated the suitability of fluorescent markers by using standard four-colour and advanced multicolour flow cytometers in relation to the effects of autofluorescence over ranges of parameters including fluorophore excitation and emission spectra, band-pass filter configurations, voltage gains and the effects of growth in the light and dark. The unicellular chlorophyte and model organism, Chlamydomonas reinhardtii, was used and findings were correlated with investigations of programmed cell death. As previously found C. reinhardtii autofluoresces in the red, far-red and infrared spectra. This is independent of laser excitation wavelength, and autofluorescence emits and spills over into detection channels of both four-colour and multicolour instruments. Band-pass filter configurations capturing longer wavelength emissions or fluorophores excited or emitted in these longer wavelengths are generally unsuitable. Furthermore, neither dark nor light incubation impacted the autofluorescent signals. Consideration of these algal autofluorescent properties and their spillover effects is required to avoid erroneous results. Recommendations for the use of a range of fluorophores in programmed cell death and other studies in C. reinhardtii using four-colour and multicolour instruments are made.     

Correction of cellular autofluorescence in flow cytometry by mathematical modeling of cellular fluorescence

A method for the correction of background fluorescence in flow cytometry with special relevance to the quantitation of low levels of cellular surface membrane antigens is presented. The method is based on the mathematical modeling of cellular fluorescence distributions of background fluorescence (autofluorescence control or irrelevant antibody control) and total fluorescence (positively stained cells). Algorithms based on two models and utilizing only the routinely available background and total fluorescence histograms are developed and implemented in computer programs. These allow estimation of the fluorescence histogram corresponding exclusively to immunofluorescence staining of the cell surface antigen of interest. Thus, the correction of background fluorescence is effected solely with software processing of routinely available data; no additional hardware or parameter determinations are necessary. Two models were chosen to be physically plausible and to represent extremes in correlation between background and probe fluorescence. Extremes were chosen to assess the solution dependence on model and to provide bounds to the actual solution when no information on correlation is available. Results are presented for both computer simulations and for an actual assay of the CR1 complement receptor on human erythrocytes to test and illustrate the technique. Alternatively, data can be tested assuming a particular model to explore the relationship, if any, between specific and nonspecific fluorescence.     

Improvement of phytoplankton culture isolation using single cell sorting by flow cytometry

Flow cytometry provides a tool to physically sort single algal cells in order to obtain clonal cultures. During sorting, cells are submitted to physical stress factors such as high fluidic pressure, exposure to the laser beam, electrostatic charges, deflection through high voltage fields, and collisions with container surfaces. All of these can damage the cells of interest and success rates for initiation of cultures from flow‐sorted cells are generally very low. We found that the addition of bovine serum albumin in the culture medium into which cells were sorted drastically improved the success of initiation of pico‐ and nano‐eukaryotic phytoplankton strains. Adding a mixture of antibiotics (Penicillin, Neomycin, Streptomycin) to the medium in order to slow down bacterial growth further improved culture development. This approach was successfully used to isolate taxonomically diverse strains, including novel taxa, from a fresh sample obtained in the English Channel and from enrichment cultures established during an Atlantic meridional transect cruise. We anticipate that these improvements will be useful to clone or purify existing cultures and to isolate novel cultures from oceanic samples.     

Assessment of bacterial viability status by flow cytometry and single cell sorting

Rapid bacterial detection and viability measurements have been greatly enhanced by recent advances in the use of fluorescent stains in cytometry. It has previously been shown that four physiological states can be distinguished : reproductively viable, metabolically active, intact and permeabilized. Previous sorting experiments have shown that not all intact cells readily grow, but some intact cells can grow even when they fail to show metabolic activity, as determined by esterase turnover. To circumvent the limitations imposed by active dye extrusion or cell dormancy on viability measurements used to date (e.g. enzyme activity or cell polarization), a fast triple fluorochrome staining procedure has been developed that takes account of these problems. This allows further cellular characterization of intact cells by : active exclusion of ethidium bromide (EB) (metabolically active cells), uptake of EB but exclusion of bis-oxonol (BOX) (de-energized but with a polarized cell membrane) and uptake of both dyes (depolarized). Permeabilized cells were identified by propidium iodide (PI) uptake. The method was validated using an electronically programmable single cell sorter (EPICS Elite^®) and aged Salmonella typhimurium cells. Reproductive viability was determined by sorting single cells to their staining pattern directly onto agar plates. Most polarized cells could be recovered as well as a significant fraction of the depolarized cells, demonstrating that depolarization is a sensitive measure of cell damage but a poor indicator of cell death.     

Dual laser flow cytometry: focal length compensation when focussing through a single lens

Dual laser operation of flow cytometers, using a single focussing lens for both beams, requires compensating for chromatic aberration of the lens. By using a prefocussing lens at a fixed position in one of the laser beams, complete focal length compensation is obtained without any loss in system performance.     

Simultaneous analysis of immunophenotype and apoptosis of murine thymocytes by single laser flow cytometry.

The study of the role of apoptosis in thymocyte development has been hampered by the lack of a means of directly immunophenotyping cells undergoing the early phase of apoptosis. This restriction has been overcome by single laser flow cytometry in which apoptosis is detected by Ethidium Bromide (EBr) staining and cell phenotype by binding of FITC-labelled antibody. The initial phase of apoptosis is observed as a cell population that stains faintly with EBr preceding the characteristically bright EBr-staining normally associated with cell death. Here we directly demonstrate using single laser flow cytometry that CD4+ CD8+ CD3low/CD3intermediate thymocytes undergo apoptosis in vitro in response to glucocorticoid treatment.     

Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting

With the increased awareness of the problems associated with the growth dependent analysis of bacterial populations, direct optical detection methods such as flow cytometry have enjoyed increased popularity over the last few years. Among the analyses discussed here are: (1) Bacterial discrimination from other particles on the basis of nucleic acid staining, using sample disaggregation to provide fast reliable enumeration while minimizing data artefacts due to post sampling growth; (2) Determination of basic cell functions such as reproductive ability, metabolic activity and membrane integrity, to characterise the physiological state or degree of viability of bacteria; and (3) The use of single cell sorting onto agar plates, microscope slides or into multi-well plates to correlate viability as determined by cell growth with fluorescent labelling techniques. Simultaneous staining with different fluorochromes provides an extremely powerful way to demonstrate culture heterogeneity, and also to understand the functional differences revealed by each stain in practical applications. Analysis of bacterial fermentations showed a considerable drop (20%) in membrane potential and integrity during the latter stages of small scale (5L), well mixed fed-batch fermentations. These changes, not found in either batch or continuous culture fermentations, are probably due to the severe, steadily increasing stress associated with glucose limitation during the fed-batch process, suggesting 'on-line' flow cytometry could improve process control. Heat injured cells can already show up to 4 log of differences in recovery in different pre-enrichment media, thus contributing to the problem of viable but non-culturable cells (VBNC's). Cytometric cell sorting demonstrated decreasing recovery with increasing loss of membrane function. However, a new medium protecting the cells from intracellular and extracellular causes of oxidative stress improved recovery considerably. Actively respiring cells showed much higher recovery improvement than the other populations, demonstrating for the first time the contribution of oxidative respiration to intracellular causes of damage as a key part of the VBNC problem. Finally, absolute and relative frequencies of one species in a complex population were determined using immunofluorescent labelling in combination with the analysis of cell function. The detail and precision of multiparameter flow cytometric measurements of cell function at the single cell level now raise questions regarding the validity of classical, growth dependent viability assessment methods.     

基于单克隆抗体的多元弧菌流式细胞术检测技术的研究

【目的】建立一种基于单克隆抗体的多元弧菌流式细胞仪检测技术。【方法】以副溶血弧菌表面蛋白r-OmpW的单克隆抗体(mAb)为基础,以细胞染色率为指标优化流式细胞仪检测副溶血弧菌时所需mAb的反应浓度和反应时间。通过菌落数对比评价在优化条件下流式细胞术方法的准确度、检出限和精密度。根据所建立的流式细胞术平台分析鉴定单抗对其它5种多元弧菌的特异性。【结果】流式细胞仪检测副溶血弧菌时所需r-OmpW单克隆抗体的优化反应浓度为20 mg/L,反应时间为60 min。当菌浓在104 107cells/mL范围时,检测值可信度较高,可特异性识别5种病原弧菌。对含不同菌体浓度的样品进行重复检测,变异系数均在7%以内。【结论】所建立的这种基于单克隆抗体的多元弧菌流式细胞仪检测技术可快速准确地检测多种病原弧菌。     

Evaluation of a green laser pointer for flow cytometry.

Flow cytometers typically incorporate expensive lasers with high-quality (TEM00) output beam structure and very stable output power, significantly increasing system cost and power requirements. Red diode lasers minimize power consumption and cost, but limit fluorophore selection. Low-cost DPSS laser pointer modules could possibly offer increased wavelength selection but presumed emission instability has limited their use. A $160 DPSS 532 nm laser pointer module was first evaluated for noise characteristics and then used as the excitation light source in a custom-built flow cytometer for the analysis of fluorescent calibration and alignment microspheres. Eight of ten modules tested were very quiet (RMS noise < or = 0.6% between 0 and 5 MHz). With a quiet laser pointer module as the light source in a slow-flow system, fluorescence measurements from alignment microspheres produced CVs of about 3.3%. Furthermore, the use of extended transit times and < or =1 mW of laser power produced both baseline resolution of all 8 peaks in a set of Rainbow microspheres, and a detection limit of <20 phycoerythrin molecules per particle. Data collected with the transit time reduced to 25 micros (in the same instrument but at 2.4 mW laser output) demonstrated a detection limit of approximately 75 phycoerythrin molecules and CVs of about 2.7%. The performance, cost, size, and power consumption of the tested laser pointer module suggests that it may be suitable for use in conventional flow cytometry, particularly if it were coupled with cytometers that support extended transit times.