Discrimination of live, anti-tuberculosis agent-injured, and dead Mycobacterium tuberculosis using flow cytometry

Flow cytometry (FCM) using propidium iodide (PI)/bis-oxonol (BOX) staining can distinguish live, dead, and sublethally injured Escherichia coli by detecting intact vs. nonintact membranes (PI) and membrane potential (BOX). However, live bacteria, especially Mycobacterium tuberculosis , are not likely to be successfully discriminated from injured bacterium by FCM when utilizing the live/dead staining agents currently on the market. As injured cell membranes have integrity like that of live cells and are regarded as such by FCM, the distinction between live and injured cells has depended on the culture method, where injured bacteria cannot grow in general. We have previously shown that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro . In this study, we found that the chromosomal DNA of antibiotic-injured, but not live, M. tuberculosis could be cleaved within 2 h by EMA, and that the resultant decrease in the spaces of DNA base pairs could greatly inhibit the intercalation of SYTO9 in FCM. The percentage value of SYTO9⁺/PI⁻ quadrant from antibiotic-injured M. tuberculosis after EMA treatment decreased by at least 80%, compared with that before EMA, but such a phenomenon did not take place in live cells. FCM (SYTO9/PI) following EMA treatment is a very rapid, simple, and effective method for discriminating live, antibiotic-injured, and dead M. tuberculosis without culture.     

Evaluation of touch-down real-time PCR based on SYBR Green I fluorescent dye for the detection of Neisseria meningitidis in clinical samples

Antibiotic treatment prior to transport or admission of patients to hospital has reduced the proportion of patients with invasive meningococcal disease (IMD) from whom Neisseria meningitidis can be isolated by standard microbiological techniques. Assays to detect the crgA gene were used to detect meningococcal DNA by both conventional polymerase chain reaction (PCR) and real-time PCR (RTPCR) in relation to microbiological diagnosis of cases over two years between 2002 and 2003. The sensitivity of both PCR assays for culture-confirmed cases was 93% and the specificity was 98.6%. Agreement between the two PCR assays was 96.2%. The inter- and intra-assay variations and effects of different amounts of DNA on the melting temperatures were examined. The touch-down RTPCR based on SYBR Green I fluorescent dye detected and characterized N. meningitidis in clinical samples within one hour.     

Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.     

Using phytoplankton and flow cytometry to analyze grazing by marine organisms

Phytoplankton can, through their autofluorescent characteristics, be thought of as tracer particles in much the same way as fluorescent microspheres when used in particle uptake experiments. Flow cytometric techniques can be used to differentiate phytoplankton from other suspended particles by the two primary autofluorescing photosynthetic pigments, chlorophyll and phycoerythrin. Based on these characteristics, phytoplankton assemblages have been used to assess grazing rates, particle selectivity, and endocytotic abilities in various marine species, from single-celled organisms to higher invertebrates.     

Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry

In this paper, we demonstrate the use of synthetic polyamide probes to fluorescently label heterochromatic regions on human chromosomes for discrimination in cytogenetic preparations and by flow cytometry. Polyamides bind to the minor groove of DNA in a sequence-specific manner. Unlike conventional sequence-specific DNA or RNA probes, polyamides can recognize their target sequence without the need to subject chromosomes to harsh denaturing conditions. For this study, we designed and synthesized a polyamide to target the TTCCA-motif repeated in the heterochromatic regions of chromosome 9, Y and 1. We demonstrate that the fluorescently labeled polyamide binds to its target sequence in both conventional cytogenetic preparations of metaphase chromosomes and suspended chromosomes without denaturation. Chromosomes 9 and Y can be discriminated and purified by flow sorting on the basis of polyamide binding and Hoechst 33258 staining. We generate chromosome 9- and Y-specific ‘paints’ from the sorted fractions. We demonstrate the utility of this technology by characterizing the sequence of an olfactory receptor gene that is duplicated on multiple chromosomes. By separating chromosome 9 from chromosomes 10–12 on the basis of polyamide fluorescence, we determine and differentiate the haplotypes of the highly similar copies of this gene on chromosomes 9 and 11.     

Strategies to minimize false positives and interpret novel microdeletions based on maternal copy-number variants in 87,000 noninvasive prenatal screens

Background

Noninvasive prenatal screening (NIPS) of common aneuploidies using cell-free DNA from maternal plasma is part of routine prenatal care and is widely used in both high-risk and low-risk patient populations. High specificity is needed for clinically acceptable positive predictive values. Maternal copy-number variants (mCNVs) have been reported as a source of false-positive aneuploidy results that compromises specificity.

Methods

We surveyed the mCNV landscape in 87,255 patients undergoing NIPS. We evaluated both previously reported and novel algorithmic strategies for mitigating the effects of mCNVs on the screen’s specificity. Further, we analyzed the frequency, length, and positional distribution of CNVs in our large dataset to investigate the curation of novel fetal microdeletions, which can be identified by NIPS but are challenging to interpret clinically.

Results

mCNVs are common, with 65% of expecting mothers harboring an autosomal CNV spanning more than 200 kb, underscoring the need for robust NIPS analysis strategies. By analyzing empirical and simulated data, we found that general, outlier-robust strategies reduce the rate of mCNV-caused false positives but not as appreciably as algorithms specifically designed to account for mCNVs. We demonstrate that large-scale tabulation of CNVs identified via routine NIPS could be clinically useful: together with the gene density of a putative microdeletion region, we show that the region’s relative tolerance to duplications versus deletions may aid the interpretation of microdeletion pathogenicity.

Conclusions

Our study thoroughly investigates a common source of NIPS false positives and demonstrates how to bypass its corrupting effects. Our findings offer insight into the interpretation of NIPS results and inform the design of NIPS algorithms suitable for use in screening in the general obstetric population.

     

Kinetics of virus adsorption to single cells using fluorescent membrane probes and multiparameter flow cytometry

Different genetic stains of avian RNA tumor virus (ATV) were labeled with the fluorescent membrane probe R-18 (rhodamine conjugated to a hydrocarbon chain) and cellular receptors for virus infection were analyzed on a rapid, single-cell basis by a multiparameter cell sorter. Chicken cells genetically susceptible to various R-18 ATV were found to adsorb much more virus, as measured by increased fluorescent binding, than did genetically resistant chicken cells. Virus binding to receptor sites could be saturated with increased concentrations of labeled virus. This binding could be altered by removal of the polycation, polybrene, indicating the important influence of electrostatic forces. Correlated time measurements of virus binding to single cells were taken with these fluorescence measurements allowing for a minute-to-minute study of the kinetics of viral adsorption to resistant and susceptible cells. The ratio of fluorescence (proportional to the number of virions bound per cell) to light scatter (proportional to cell surface area) on a cell-to-cell basis was analyzed to examine the heterogeneity in fluorescent virion bound per unit cell surface area within a given cell type. With these calculations, it was found that a large amount, but not all, of observed fluorescence heterogeneity merely reflects differences in cell surface areas. However, there are significant differences in viral receptor site densities within this supposedly homogeneous population of cells. This study represents a successful application of fluorescent membrane probes and flow cytometry to the study of cellular responses to viral infection at the single-cell level. Sine large numbers of cells can be examined rapidly, small subpopulations of live virally susceptible or resistant cells can be cloned by multiparameter cell sorting.     

Flow cytometry as a tool to identify Mycobacterium tuberculosis interaction with the immune system and drug susceptibility

Flow cytometric analysis is a useful and widely employed tool to identify immunological alterations caused by different microorganisms, including Mycobacterium tuberculosis. However, this tool can be used for several others analysis. We will discuss some applications for flow cytometry to the study of M. tuberculosis, mainly on cell surface antigens, mycobacterial secreted proteins, their interaction with the immune system using inflammatory cells recovered from peripheral blood, alveolar and pleura spaces and the influence of M. tuberculosis on apoptosis, and finally the rapid determination of drug susceptibility. All of these examples highlight the usefulness of flow cytometry in the study of M. tuberculosis infection.     

Plasmodium falciparum: Development and validation of a measure of intraerythrocytic growth using SYBR Green I in a flow cytometer

Reliable analytical techniques to test growth-promoting and antimalarial efficacy on plasmodia are very important. Flow cytometry (FCM) offers the possibility to study developmental stages of intraerythrocytic growth of malaria parasites using nucleic acid staining. To analyze the growth of Plasmodium falciparum SYBR Green I was introduced as an intercalating dye with FCM for the 488 nm line of an argon laser. Procedures employing FCM, including fixatives, dye concentrations, dilution buffer, and staining period, were optimized to simplify the method. FCM as described here allows parasitemia and parasites of different stages to be quantified according to the DNA content. The proportion of parasitized erythrocytes estimated by FCM and the Giemsa method agreed with determination by parasite lactate dehydrogenase. The protocol was extended to merozoite counting as a sensitive assay of growth inhibition of the parasite.     

Using flow cytometry to estimate pollen DNA content: improved methodology and applications

Background and Aims

Flow cytometry has been used to measure nuclear DNA content in pollen, mostly to understand pollen development and detect unreduced gametes. Published data have not always met the high-quality standards required for some applications, in part due to difficulties inherent in the extraction of nuclei. Here we describe a simple and relatively novel method for extracting pollen nuclei, involving the bursting of pollen through a nylon mesh, compare it with other methods and demonstrate its broad applicability and utility.

Methods

The method was tested across 80 species, 64 genera and 33 families, and the data were evaluated using established criteria for estimating genome size and analysing cell cycle. Filter bursting was directly compared with chopping in five species, yields were compared with published values for sonicated samples, and the method was applied by comparing genome size estimates for leaf and pollen nuclei in six species.

Key Results

Data quality met generally applied standards for estimating genome size in 81 % of species and the higher best practice standards for cell cycle analysis in 51 %. In 41 % of species we met the most stringent criterion of screening 10 000 pollen grains per sample. In direct comparison with two chopping techniques, our method produced better quality histograms with consistently higher nuclei yields, and yields were higher than previously published results for sonication. In three binucleate and three trinucleate species we found that pollen-based genome size estimates differed from leaf tissue estimates by 1·5 % or less when 1C pollen nuclei were used, while estimates from 2C generative nuclei differed from leaf estimates by up to 2·5 %.

Conclusions

The high success rate, ease of use and wide applicability of the filter bursting method show that this method can facilitate the use of pollen for estimating genome size and dramatically improve unreduced pollen production estimation with flow cytometry.     

Enumeration and Cell Cycle Analysis of Natural Populations of Marine Picoplankton by Flow Cytometry Using the Nucleic Acid Stain SYBR Green I

The novel dye SYBR Green I binds specifically to nucleic acids and can be excited by blue light (488-nm wavelength). Cell concentrations of prokaryotes measured in marine samples with this dye on a low-cost compact flow cytometer are comparable to those obtained with the UV-excited stain Hoechst 33342 (bis-benzimide) on an expensive flow cytometer with a water-cooled laser. In contrast to TOTO-1 and TO-PRO-1, SYBR Green I has the advantage of clearly discriminating both heterotrophic bacteria and autotrophic Prochlorococcus cells, even in oligotrophic waters. As with TOTO-1 and TO-PRO-1, two groups of heterotrophic bacteria (B-I and B-II-like types) can be distinguished. Moreover, the resolution of DNA distribution obtained with SYBR Green I is similar to that obtained with Hoechst 33342 and permits the analysis of the cell cycle of photosynthetic prokaryotes over the whole water column.     

Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria.

Current limitations in the methodology for enumeration and identification of airborne bacteria compromise the precision and accuracy of bioaerosol exposure assessment. In this study, flow cytometry and fluorescent in situ hybridization (FISH) were evaluated for the assessment of exposures to airborne bacteria. Laboratory-generated two-component bioaerosols in exposures chambers and complex native bioaerosols in swine barns were sampled with two types of liquid impingers (all-glass impinger-30 and May 3-stage impinger). Aliquots of collection media were processed and enumerated by a standard culture technique, microscopy, or flow cytometry after nucleic acid staining with 4',6-diamidino-2-phenylindole (DAPI) and identified taxonomically by FISH. DAPI-labeled impinger samples yielded comparable estimates of bioaerosol concentrations when enumerated by microscopy or flow cytometry. The standard culture method underestimated bioaerosol concentrations by 2 orders of magnitude when compared to microscopy or flow cytometry. In the FISH method, aliquots of collection media were incubated with a probe universally complementary to eubacteria, a probe specific for several Pseudomonas species, and a probe complementary to eubacteria for detection of nonspecific binding. With these probes, FISH allowed quantitative identification of Pseudomonas aeruginosa and Escherichia coli bioaerosols in the exposure chamber without measurable nonspecific binding. Impinger samples from the swine barn demonstrated the efficacy of the FISH method for the identification of eubacteria in a complex organic dust. This work demonstrates the potential of emerging molecular techniques to complement traditional methods of bioaerosol exposure assessment.     

Use of flow cytometry in an apoptosis assay to determine pH and temperature stability of shiga-like toxin 1

Shiga toxins and Shiga-like toxins (Stx) are a relatively large group of cytotoxins produced by certain serotypes of Shigella and E. coli (STEC). These toxins are responsible for diarrhea, hemorrhagic colitis and may induce hemolytic uremic syndrome (HUS) with serious consequences in young children. The toxins are proteins made up of 5 small B subunits responsible for binding to an outer membrane ligand on host cells and surround the larger, biologically active A subunit. For Shiga-like toxin 1 (Stx1), the cellular receptor is the carbohydrate globotriose. Stx1was purified from STEC. We utilized induction of apoptosis in the human monocyte cell line THP-1, as a biological endpoint to test the stability of Stx1 activity added to fruit punch at different pH (2-9) and temperatures (4 and 20 degrees C). A flow cytometric method was used to test for early and late apoptotic events based on binding of R-phycoerytherin-labeled annexin V to exposed membrane phosphatidyl serine. Membrane permeability to 7-Amino-actinomycin corresponds with late apoptosis or necrosis. The combination of acid pH and higher storage temperature resulted in greatest degree of toxin inactivation. This approach provides a rapid and high throughput method to determine the functional activity of Stx1, and related toxins in a food matrix.     

Highly fluorescent GFPm2+‐based genome integration‐proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages

Study of activity of cloned promoters in slow‐growing Mycobacterium tuberculosis during long‐term growth conditions in vitro or inside macrophages, requires a genome‐integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate‐independent, easily scorable and highly sensitive reporter gene. In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon‐optimized gfp_m²⁺ gene, coding for GFP_m²⁺ of highest fluorescence reported till date, mycobacteriophage L5 attP‐int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFP_m²⁺ from M. tuberculosis and M. smegmatis genome. Expression of GFP_m²⁺, driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2‐promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. Stable expression from genome‐integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long‐term in vitro growth or stress conditions, or inside macrophages.     

Major histocompatibility complex class I and II expression on macrophages containing a virulent strain of Brucella abortus measured using green fluorescent protein-expressing brucellae and flow cytometry

Immune responses appropriate for control of an intracellular pathogen are generated in mice infected with Brucella abortus, shown by the ability of T cells to adoptively transfer resistance to naive mice. The infection nevertheless persists for months. It was hypothesized that one factor in maintaining the infection despite the presence of immune T cells was suboptimal expression of major histocompatibility complex (MHC) molecules on macrophages containing brucellae. This would allow B. abortus to elude detection by the host's immune system. To test this, B. abortus organisms expressing green fluorescent protein (GFP-Brucella) were constructed and three-color flow cytometry used to evaluate MHC expression on macrophages following in vitro or in vivo infection. When infected in vitro, the levels of MHC class I and class II expression on J774 macrophages containing GFP-Brucella were the same or higher than on macrophages without GFP-Brucella in the same cultures. Similarly, the MHC expression was higher on GFP(+) peritoneal exudate cells following infection or phagocytosis of heat-killed GFP-Brucella than it was on uninfected peritoneal exudate cells. Following in vivo infection of mice the level of MHC class I and II expression on GFP(+) cells in their spleens (the main site of infection) also tended to be as high as or higher than that on the GFP-negative cells. The only in vivo GFP(+) cells that showed a decreased MHC expression was a population of splenic Mac1(+) cells recovered from interferon-gamma gene-disrupted mice at the time of their death due to an overwhelming number of bacteria per spleen. Overall, it was concluded that decreased MHC expression is not a general principle associated with brucella infection of macrophages and thus not likely to contribute to maintenance of the chronic infection.     

Malignancy index based on flow cytometry and histology for renal cell carcinomas and its correlation to prognosis

Fifty-five human renal cell carcinomas, removed by nephrectomy, were classified using flow cytometry and histology. The parameters obtained by flow cytometry were DNA index, fractions of cells in the phases, and fractions of tumor cells versus normal cells. From histology, the routine classification of tumor structure and morphology were obtained, and a nuclear grading according to Arner et al. (Acta Chir Scand [Suppl] 346:1-12, 1965) was determined. All parameters including tumor stage according to Robson et al. (J Urol 101:297-301, 1969) were subjected to discriminant analysis in order to define a malignancy index. The patients were divided into two groups with good and poor prognosis (low and high risk). Those patients who were free of multiple metastases in 2 years and more after nephrectomy were assigned to the group "good prognosis"; those who developed multiple metastases for 2 years represented the "poor prognosis" group. The malignancy index determined by discriminant analysis yielded 91% correct predictions of prognosis.