Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

ABSTRACT: BACKGROUND: Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. METHODS: Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. RESULTS: A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signalbackground improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. DISCUSSION: Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene-expression profiling and analysis. The approach outlined here results in substantially improved yield of GFP-expressing parasites, and requires decreased sorting time in comparison to standard methods. It is anticipated that this protocol will be useful for a wide range of applications involving rare events.     

Evaluation of flow cytometry as a method for quantification of circulating blood cell populations in salmonid fish

Flow cytometry was used to estimate the proportions of different blood cell types in brown and rainbow trout. On the basis of forward light scatter and 90° side scatter three populations were differentiated. The relative abundance of these cells correlated with that of erythrocytc (r²= 0.994), lymphocyte plus thrombocyte(r²= 0.676) and neutrophil populations (r²= 0.571) enumerated by direct microscopy. By density gradient separation of cells, cell sorting and acridine orange staining it was confirmed that these cell types could be assigned to the populations detected. Changes in blood cell populations were monitored by flow cytometry in a group of experimental fish placed under confinement stress. Flow cytometry proved to be a rapid and reliable method for monitoring cell population dynamics in fish blood.     

A low-cost flow cytometric assay for the detection and quantification of apoptosis using an anionic halogenated fluorescein dye

We describe here a technical improvement of an established colorimetric method used to detect and measure the occurrence of apoptosis in mammalian cells during in vitro cell culture. This assay uses an anionic halogenated fluorescein dye that is taken up by apoptotic cells at the stage of phosphatidylserine externalization. We demonstrate that apoptotic cells stained with this dye can be detected by flow cytometric analysis. Furthermore, we show that the modified method compares well with the standard annexin-V-based apoptosis assay and that it is significantly more cost-effective than the annexin-V assay.     

A rapid and sensitive flow cytometric method for the detection of multidrug-resistant cells

Multidrug-resistant (MDR) cells are characterized by a defect in drug accumulation caused by activity of an energy-dependent rapid drug efflux pump. The action of this drug pump can be inhibited by specific agents, referred to as membrane transport modulating agents (MTMAs), resulting in a restoration of the intracellular drug accumulation. This paper presents a flow cytometric assay for the detection of MDR cells, which is based on the ability of these cells to respond to MTMAs. Daunorubicin net-uptake kinetics were measured of anthracycline-sensitive (A2780/S) and -resistant (A2780/R) human ovarian carcinoma cells in vitro. A2780/R cells accumulated significantly less (about a factor of 5) daunorubicin as compared to A2780/S cells. Addition of verapamil or cyclosporin A to A2780/R cells at steady-state daunorubicin uptake led to a dose-dependent increase in cellular daunorubicin accumulation. The sensitivity of the assay was determined by testing mixtures of A2780/S and A2780/R cells. Analysis of A2780/S cells contaminated with A2780/R cells showed that as few as 2.5% MDR cells could readily be detected in the mixture. In conclusion, this functional assay enables the detection of MDR cells in a heterogeneous cell suspension and is ideally suited for the study of the occurrence of typical MDR in human cancer.     

Establish a flow cytometric method for quantitative detection of Beclin-1 expression

Flow cytometry is an advanced technology for efficient, rapid, specific and multi-parameter analysis of single cells in various basic research fields including cytobiology, immunology, genetic, hematology and other basic research. Beclin-1 protein is an important indicator in monitoring autophagic activity. However, quantitative flow cytometry had been rarely reported till now to be applied in the detection of Beclin-1 expression. The present study was aimed to establish a flow cytometric method for quantitative detection of Beclin-1 expression by employing the autophagy inhibitor 3-methyladenine as the control. A multi-parameter optimal method for Beclin-1 protein staining is as follows. 2 % bovine serum albumin in phosphate buffered saline was used for sample block. Concentration of primary antibody was 0.004 μg/μL. Samples were incubated at room temperature (25 °C) for 30 min. The prepared samples had better to be detected immediately or to be stored at 4 °C and detected within 6 h, otherwise the samples should be fixed in 1 % paraformaldehyde storing at 4 °C and detected within 3 d. Furthermore, we employed the immunohistochemistry to validate the method in vivo, the results confirmed flow cytometric method. The established flow cytometric analysis for Beclin-1 protein has the advantage of simpleness, speediness, sensitivity and reproducibility.     

Method for flow cytometric detection of Listeria monocytogenes in milk.

This report describes a method for the detection of Listeria monocytogenes in raw milk by flow cytometric analysis of fluorescently labeled bacterial populations. The use of immunofluorescence in combination with measures of DNA content by propidium iodide labeling and size by light scattering enabled specific identification of L. monocytogenes from Streptococcus faecalis, Streptococcus agalactiae, Streptococcus uberis, Staphylococcus epidermidis, and Staphylococcus hyicus. Additional specific resolution of L. monocytogenes populations was achieved through selective enrichment of raw milk in Listeria enrichment broth. These procedures should permit the rapid screening of milk and other food samples for L. monocytogenes and eliminate many of the short-comings associated with conventional fluorescent-antibody procedures.     

A rapid method for flow cytometric cell counting and cell cycle analysis from monolayer cultures of tumor cells.

A rapid, simple, and reliable flow cytometric method using the histochemical fluorescent stain Hoechst 33342 in presence of the non-ionic detergent Triton X-100 has been reported. The processing of melanoma cell cultures to get nuclei stained with the fluorescent dye was accomplished in one step and within an hour permitted concurrent flow cytometric measurement of cell density and cell cycle analysis. The preparation is stable for more than three weeks at room temperature for flow cytometry. The histograms are reproducible and exhibit a coefficient of variation of less than 2.5% (G1 peak). The cell density measurements varied within +/- 5% limits.     

Flow cytometric quantification of apoptotic and proliferating cells applying an improved method for dissociation of spheroids

Spheroids are a promising tool for many cell culture applications, but their microscopic analysis is limited. Flow cytometry on a single cell basis, which requires a gentle but also efficient dissociation of spheroids, could be an alternative analysis. Mono-culture and coculture spheroids consisting of human fibroblasts and human endothelial cells were generated by the liquid overlay technique and were dissociated using AccuMax as a dissociation agent combined with gentle mechanical forces. This study aimed to quantify the number of apoptotic and proliferative cells. We were able to dissociate spheroids of differing size, age, and cellular composition in a single-step dissociation protocol within 10 min. The number of single cells was higher than 95% and in most cases, the viability of the cells after dissociation was higher than 85%. Coculture spheroids exhibited a higher sensitivity as shown by lower viability, higher amount of cellular debris, and a higher amount of apoptotic cells. Considerable expression of the proliferation marker Ki67 could only be seen in 1-day-old spheroids but was already downregulated on Day 3. In summary, our dissociation protocol enabled a fast and gentle dissociation of spheroids for the subsequent flow cytometric analysis. The chosen cell type had a strong influence on cell viability and apoptosis. Initially high rates of proliferative cells decreased rapidly and reached values of healthy tissue 3 days after generation of the spheroids. In conclusion, the flow cytometry of dissociated spheroids could be a promising analytical tool, which could be ideally combined with microscopic techniques.     

An original approach for quantification of blood vessels on the whole tumour section.

Relative abundance of tumour angiogenesis has been shown to be of clinical relevance in cancers of various locations such as the ovary. Nevertheless, several problems are encountered when quantifying tumour microvessels: (i) as many other tumour markers, vascularity pattern is often heterogeneous within the tumour mass and even within the same histological section. As a consequence, an adequate acquisition method must be developed for accurate field sampling. (ii) Manual microvessel counting is long, tedious and subject to poor reproducibility. Introduction in routine practice requires a fast, reproducible and reliable automatic image processing. In this study we present an original procedure combining a slide scanner image acquisition and a fully automatic image analysis sequence. The slide scanner offers the advantage of recording an image of the whole histological section for subsequent automatic blood vessel detection and hot spot area location. Microvessel density and surface fraction were measured for the whole section as well as within hot spots. Different immunostaining methods were tested in order to optimise the procedure. Moreover, the method proposed was submitted to a quality control procedure, with reference to interactive identification of microvessels at scanner level. This experiment showed that 93 to 97% of blood vessels were detected, according to the staining protocol used. Colour figures can be viewed on http://www.esacp.org/acp/2003/25-2/kim.htm.     

Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood.

This article explores the feasibility of the use of automated microscopy and image analysis to detect the presence of rare fetal nucleated red blood cells (NRBCs) circulating in maternal blood. The rationales for enrichment and for automated image analysis for "rare-event" detection are reviewed. We also describe the application of automated image analysis to 42 maternal blood samples, using a protocol consisting of one-step enrichment followed by immunocytochemical staining for fetal hemoglobin (HbF) and FISH for X- and Y-chromosomal sequences. Automated image analysis consisted of multimode microscopy and subsequent visual evaluation of image memories containing the selected objects. The FISH results were compared with the results of conventional karyotyping of the chorionic villi. By use of manual screening, 43% of the slides were found to be positive (>=1 NRBC), with a mean number of 11 NRBCs (range 1-40). By automated microscopy, 52% were positive, with on average 17 NRBCs (range 1-111). There was a good correlation between both manual and automated screening, but the NRBC yield from automated image analysis was found to be superior to that from manual screening (P=.0443), particularly when the NRBC count was >15. Seven (64%) of 11 XY fetuses were correctly diagnosed by FISH analysis of automatically detected cells, and all discrepancies were restricted to the lower cell-count range. We believe that automated microscopy and image analysis reduce the screening workload, are more sensitive than manual evaluation, and can be used to detect rare HbF-containing NRBCs in maternal blood.     

Simple method for quantification of fast plasma membrane movements.

We present a method for the quantification of the fast plasma membrane movements that are involved in ruffling, blebbing, fast shape change, and fast translocation. The method is based on the Kontron Vidas image analysis computer program. Video images from cells viewed through an inverted microscope were transmitted to the computer. The procedure was as follows: 4 consecutive video images were averaged (image 1); 28 s later a second set of 4 video images was averaged (image 2); image 2 was subtracted from image 1 and the grey level of each pixel of the resulting image was increased with 128 grey level units, resulting in the subtraction image, showing a uniform grey background speckled with brighter and darker spots corresponding to areas of movement. These spots were discriminated and turned into white objects against a black background. Interactive editing was used to delete artefacts that resulted from floating debris. The total area of the discriminated objects was measured, and the parameter motile area in micron2 per cell was calculated. We have applied our method to the study of motility induced in epithelial cell lines by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate and by epidermal growth factor.     

A rapid analytical technique for flow cytometric analysis of cell viability using calcofluor white M2R

Analysis of dead versus live cells is shown to be possible using Calcoflour White M2R (CFW), a fluorescent brightener. Comparison of CFW with both propidium iodide (PI) and fluorescein diacetate (FDA) was performed on a FACS 440 dual laser flow cytometer on several populations of cultured rat and mouse cell lines, peripheral leukocytes, splenocytes, diatoms, and plant protoplasts. As a measure of cell viability, staining results with CFW were strongly associated with PI (correlation coefficient of 0.9886) and FDA (inverse correlation coefficient of 0.9647). With plant and algal cells, controls are necessary as CFW does stain live cells to some extent. CFW (excitation: UV, emission max: 435 nm) can be used in conjunction with two-color immunofluorescence analysis using fluorochromes excited at 488 nm with no interference.     

Novel method for cell debris removal in the flow cytometric cell cycle analysis using carboxy-fluorescein diacetate succinimidyl ester.

BACKGROUND: Cell cycle analysis with flow cytometry using propidium iodide (PI) can be difficult in some cases because of the cell debris. Here, we introduce debris removal using intranuclear protein staining (DRIPS), a novel method for separating intact nuclei and cell debris to different populations using carboxy-fluorescein diacetate succinimidyl ester (CFSE). METHODS: To study the apoptosis-sensitivity, chicken DT40 B cell lymphoma cell line was gamma irradiated. After the irradiation, the cells were incubated up to 8 h and the stages of the cell cycle were followed with flow cytometry. RESULTS: CFSE staining, done simultaneously with PI, stained the cell debris brighter than intact nuclei and could be excluded from the histogram with a simple gating procedure. The method is reliable and reproducible and can be executed within 15 min. CONCLUSIONS: DRIPS-method greatly enhances the analysis of difficult cell cycle samples.     

Phase tracking: an improved phase detection technique for cell membrane capacitance measurements.

We describe here a technique called phase tracking that greatly improves the accuracy of measurements of the membrane capacitance of single cells. We have modified the original phase detection technique to include a method for creating calibrated changes in the resistance in series with the cell. This provides a method to automate the adjustment of the phase detector to the appropriate phase angle for measuring membrane capacitance. The phase determination depends only on the cell's electrical parameters and does not require matching of the cell impedance with that of the slow capacitance cancellation circuitry of the patch-clamp amplifier. We show here that phase tracking can accurately locate the phase of the capacitance signal and can keep the detector aligned with this signal during measurements of exocytosis in mast cells, irrespective of the large drifts which occur in cell membrane resistance, membrane capacitance, or series resistance. The phase tracking technique is a valuable tool for quantifying exocytosis and endocytosis in single cells.     

Identification of Echovirus 1 and coxsackievirus A9 receptor molecules via a novel flow cytometric quantification method

BACKGROUND: Virus-receptor binding is an essential step in every virus infectious process. Many viruses employ more than one receptor molecule or even receptor complexes for attachment. In this study, we investigate the binding of Echovirus 1 (Echo1) and Coxsackievirus A9 (CAV-9) on cell surface molecules. CAV-9 has been reported to utilize integrin alpha v beta(3) in binding to cells, whereas Echo1 has been known to utilize integrin alpha 2 beta(1). METHODS AND RESULTS We directly test whether the presence of these molecules alone was sufficient for virus binding. We devised a novel flow cytometric binding assay that enables us to quantify virus particles bound on host cells and to further determine the extent to which viruses utilize specific receptors. CONCLUSIONS: By quantifying virus particles and possible receptor molecules, we found that Echo1 utilizes mainly integrin alpha 2 beta(1). CAV-9 utilizes integrin alpha v beta(3) to a much lesser extent (40%), indicating that CAV-9 also utilizes other receptor(s).     

A flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk

AIMS: The present study describes a flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk according to the main cause of elevated counts. METHODS AND RESULTS: A total of 75 Danish bulk tank milk samples exceeding the grading level of 3.0 x 10(4) CFU ml(-1) were examined by both flow cytometry and traditional microbiological analyses. The correlation coefficient (r) between the two methods was 0.71. For the differential analyses of the dominant bacterial populations four different parameters were used to give a species-characteristic pattern. The four parameters were as follows: staining with Oregon Green conjugated wheat germ agglutinin that binds to the cell wall of bacteria, staining with hexidium iodide that binds to all bacterial DNA, the flow cytometric forward scatter and the flow cytometric side scatter. Three regions in the flow cytometric plot were defined: region 1 includes bacteria mainly associated with poor hygiene, region 2 includes psychrotrophic hygiene bacteria and region 3 includes bacteria mainly related to mastitis. The ability of the flow cytometric technique to predict the main cause of elevated bacterial counts on routine samples was examined. Comparing these results with results obtained by traditional microbiological analyses for identification showed that for 81% of the samples the two techniques agreed on the main cause of an elevated bacterial count. CONCLUSIONS: The ability of the presented flow cytometric technique to enumerate and differentiate bacteria in bulk tank milk according to the main cause of elevated counts was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: This study described the first step in development of a technique suitable for routine analyses of bulk tank milk samples. A technique indicating the main cause of an elevated count will enable the farmer to eliminate the contamination source within a short time limit.     

MTT方法评价微生物细胞活性的探讨

对MTT比色法用于评价微生物细胞活性进行了探讨。本文以大肠杆菌为模式菌株,研究了不同浓度MTT、不同用量、在不同时间对试验结果OD570值的影响,结果表明细菌数在4.9×107－4.9×108个/mL范围内测出的OD570值与细菌浓度呈良好的正相关,0.5 mg/mL MTT用量20 L,反应时间20 min时效果最佳,其相关回归方程为y = 0.1769x + 0.03,R2 = 0.9983。     

A rapid, simple and sensitive flow cytometric system for detection of Plasmodium falciparum.

We have established a rapid, simple and sensitive flow cytometric system for the detection of Plasmodium falciparum that involves lysing erythrocytes and staining parasites at the same time using a newly developed hemolysing and staining solution containing dodecyl methyl ammonium chloride and acridine orange. In this system, freed parasites of P. falciparum could be plotted separately from erythrocyte ghosts, white blood cells and platelets on the two-dimensional scattergram of forward-angle light scatter and green fluorescence by flow cytometry with an argon laser. It took only 2–3 min per sample to obtain the scattergram and analyze the data, including the time of sample preparation for flow cytometric analysis. Sample preparation with this method does not require any difficult handling procedures. The threshold of parasite detection was almost equal to that of microscopic examination for cultured P. falciparum. The results of drug-susceptibility assays using this system were also almost identical to those obtained using microscopic examination. In this system, parasites at different erythrocytic stages could be easily distinguished. This system must prove useful and practical for basic laboratory studies of P. falciparum including those requiring the differential measurement of parasites at specific erythrocytic stages.