A method to rank order water soluble compounds according to their toxicity using Caenorhabditis elegans, a Complex Object Parametric Analyzer and Sorter, and axenic liquid media

Caenorhabditis elegans (L1s) were exposed to (in order of decreasing toxicity) sodium arsenite, sodium fluoride, caffeine, valproic acid, sodium borate, or dimethyl sulfoxide in C. elegans habitation medium (CeHM) for 72 consecutive hours. At this time point nematode growth and development were assessed using a Complex Object Parametric Analyzer and Sorter (COPAS ™ ). The COPAS generated biomarkers of growth (time of flight (TOF) - a measure of axial length) and development (extinction (EXT) - a measure of optical density) were subse- quently utilized to rank compounds according to their relative toxicity, as measured by the rat oral LD-50, using artificial neural network methods. Neural network methods were utilized to analyze this data because of their ability to model nonlinear endpoints and a multilayer perceptron neural network method was used because of its capability to function well in the presence of collinearity. Using a neural network approach we found that the LD-50 was correctly predicted 96% of the time. The present study demonstrates that neural network methods can be utilized to rank compounds according to their relative toxicity using COPAS-generated data (TOF and EXT) obtained from exposing a large number of nematodes to water-soluble compounds

The Complex Object Parametric Analyzer and Sorter (COPAS) is a large particle flow cytometer designed for analyzing, sorting, and dispensing objects of varying sizes. We explored the potential of using this instrument to analyze and sort various developmental stages and egg viability of Globodera pallida. Cysts were successfully examined and sorted from debris by optimizing side-scatter and red-fluorescence parameters on the COPAS. We were able to separate eggs and second-stage juveniles from samples of mixed population using extinction and time of flight. Separation of live and dead eggs was examined following staining eggs with SYTOX Green and application of time of flight and green peak height. Data were compared with a commonly used viability assay by which eggs were stained with Meldola's Blue and examined by a microscope. COPAS proved to be effective in assessing viability by detecting two separate gates: live eggs having green fluorescence peaks <190 and dead eggs with the peaks >190. The application of COPAS in combination with SYTOX Green detected a greater number of live eggs than the Meldola's assay, suggesting that SYTOX Green provided an overestimate of live eggs. COPAS noticeably increased the accuracy and reduced the time required for screening and analyzing nematode populations.

Complex Object Parametric Analyzer and Sorter (COPAS) devices are large-object, fluorescence-capable flow cytometers used for high-throughput analysis of live model organisms, including Drosophila melanogaster, Caenorhabditis elegans, and zebrafish. The COPAS is especially useful in C. elegans high-throughput genome-wide RNA interference (RNAi) screens that utilize fluorescent reporters. However, analysis of data from such screens is relatively labor-intensive and time-consuming. Currently, there are no computational tools available to facilitate high-throughput analysis of COPAS data. We used MATLAB to develop algorithms (COPAquant, COPAmulti, and COPAcompare) to analyze different types of COPAS data. COPAquant reads single-sample files, filters and extracts values and value ratios for each file, and then returns a summary of the data. COPAmulti reads 96-well autosampling files generated with the ReFLX adapter, performs sample filtering, graphs features across both wells and plates, performs some common statistical measures for hit identification, and outputs results in graphical formats. COPAcompare performs a correlation analysis between replicate 96-well plates. For many parameters, thresholds may be defined through a simple graphical user interface (GUI), allowing our algorithms to meet a variety of screening applications. In a screen for regulators of stress-inducible GFP expression, COPAquant dramatically accelerated data analysis and allowed us to rapidly move from raw data to hit identification. Because the COPAS file structure is standardized and our MATLAB code is freely available, our algorithms should be extremely useful for analysis of COPAS data from multiple platforms and organisms. The MATLAB code is freely available at our web site (www.med.upenn.edu/lamitinalab/downloads.shtml).

This protocol describes a procedure for screening small molecules for bioactivity and a genetic approach to target identification using the nematode Caenorhabditis elegans as a model system. Libraries of small molecules are screened in 24-well plates that contain a solid agar substrate. On top of the agar mixture, one small-molecule species is deposited into each well, along with worm food (E. coli), and two third-stage or fourth-stage larval worms using a COPAS (Complex Object Parametric Analyzer and Sorter) Biosort. Three to five days later the plates are screened for phenotype. Images of the wells are acquired and archived using a HiDI 2100 automated imaging system (Elegenics). Up to 2,400 chemicals can be screened per week. To identify the predicted protein target of a bioactive molecule, wild-type worms are mutagenized using ethylmethanesulfonate (EMS). Progeny are screened for individuals resistant to the molecules effects. The candidate mutant target that confers resistance is then identified. Target identification might take months.

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.

Transgenic mosquitoes are used in many aspects of mosquito research. First, they can help answer biological questions to advance scientific knowledge-for example, in the fields of mosquito-pathogen interactions, insect immunity, or olfaction. Second, transgenic technologies may be used to develop much needed novel vector control strategies, such as mosquitoes that are unable to transmit disease or transgenes that sterilize mosquito females to suppress vector populations. Here, we introduce how researchers use various selection markers to screen for transgenic mosquito larvae following a transgenesis experiment. Common procedures include using a binocular fluorescence microscope for initial screening. For higher-throughput screening, a flow cytometer known as Complex Object Parametric Analyzer and Sorter (COPAS) can be used to stabilize transgenic lines through the purification of homozygous individuals or to manage transgene frequency in established transgenic lines. In particular, COPAS sorting allows the production of mosquito larval cultures composed of a mixture of genotypes (control and genetically modified larvae) with the goal of raising both groups of mosquitoes under the same environmental conditions in preparation for a controlled phenotype assessment. It can also be used to produce large populations of male mosquitoes, which should facilitate the development of mosquito control intervention strategies similar to the sterile insect technique (SIT), which aims to release large numbers of sterile males that will mate with and sterilize wild females to suppress mosquito populations. Finally, the utilization of a puromycin resistance marker cassette to screen for transgenic Anopheles larvae is also introduced.

Studying the molecular regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for understanding how the kidney contributes to blood pressure regulation. Until now, a native mammalian cell model to investigate this transporter remained unknown. Our aim here is to establish, for the first time, a primary distal convoluted tubule (DCT) cell culture exhibiting transcellular thiazide-sensitive Na(+) transport. Because parvalbumin (PV) is primarily expressed in the DCT, where it colocalizes with NCC, kidneys from mice expressing enhanced green-fluorescent protein (eGFP) under the PV gene promoter (PV-eGFP-mice) were employed. The Complex Object Parametric Analyzer and Sorter (COPAS) was used to sort fluorescent PV-positive tubules from these kidneys, which were then seeded onto permeable supports. After 6 days, DCT cell monolayers developed transepithelial resistance values of 630 ± 33 Ω·cm(2). The monolayers also established opposing transcellular concentration gradients of Na(+) and K(+). Radioactive (22)Na(+) flux experiments showed a net apical-to-basolateral thiazide-sensitive Na(+) transport across the monolayers. Both hypotonic low-chloride medium and 1 μM angiotensin II increased this (22)Na(+) transport significantly by four times, which could be totally blocked by 100 μM hydrochlorothiazide. Angiotensin II-stimulated (22)Na(+) transport was also inhibited by 1 μM losartan. Furthermore, NCC present in the DCT monolayers was detected by immunoblot and immunocytochemistry studies. In conclusion, a murine primary DCT culture was established which expresses functional thiazide-sensitive Na(+)-Cl(-) transport.

Potato cyst nematode (PCN) cysts consist of heterogenous populations of eggs, juveniles, and eggshells that make manual sorting of individual life stages cumbersome. The number of viable PCN eggs is a major determinant of crop damage. An accurate high-throughput PCN egg viability assay is useful for developing effective management and eradication plans. In this study, we present a method for rapid and precise enumeration and sorting of PCN eggs and juveniles, along with an egg viability assessment by staining eggs with the fluorescent stain, acridine orange, and sorting with the Complex Object Parametric Analyzer and Sorter (COPAS) system, a large particle flow cytometer. Both size sorting and fluorescent sorting capabilities of the COPAS were explored. By using the COPAS, sorting efficiency for eggs and preparasitic second-stage juveniles (J2s) was 97.6 and 97.2%, respectively, with 99% recovery at a flow rate of 15 events/s. Purity of sorted live and dead eggs was 95.5 and 94.1%, respectively. Sorting of J2s by size indicated that 15 to 16.4% of Globodera ellingtonae or G. pallida had an average body length of 436.1 ± 3.4 µm compared with an average size of 512.9 ± 4.4 µm for the majority of the J2 population for both species. A red autofluorescing J2 population was also identified through sorting. Sorting of eggs by flow cytometry did not significantly affect hatching (55.1 ± 1.2 and 53.9 ± 1.6%, respectively, for sorted or nonsorted eggs) or juvenile motility (91.3 ± 1.0 or 90.1 ± 1.1%, respectively), thus confirming that the method does not impair the biological activity of the nematode.

The fungus Aspergillus niger forms (sub)millimeter microcolonies within a liquid shaken culture. Here, we show that such microcolonies are heterogeneous with respect to size and gene expression. Microcolonies of strains expressing green fluorescent protein (GFP) from the promoter of the glucoamlyase gene glaA or the ferulic acid esterase gene faeA were sorted on the basis of diameter and fluorescence using the Complex Object Parametric Analyzer and Sorter (COPAS) technology. Statistical analysis revealed that the liquid shaken culture consisted of two populations of microcolonies that differ by 90 μm in diameter. The population of small microcolonies of strains expressing GFP from the glaA or faeA promoter comprised 39% and 25% of the culture, respectively. Two populations of microcolonies could also be distinguished when the expression of GFP in these strains was analyzed. The population expressing a low level of GFP consisted of 68% and 44% of the culture, respectively. We also show that mRNA accumulation is heterogeneous within microcolonies of A. niger. Central and peripheral parts of the mycelium were isolated with laser microdissection and pressure catapulting (LMPC), and RNA from these samples was used for quantitative PCR analysis. This analysis showed that the RNA content per hypha was about 45 times higher at the periphery than in the center of the microcolony. Our data imply that the protein production of A. niger can be improved in industrial fermentations by reducing the heterogeneity within the culture.

Transgenic mosquitoes are widely used in mosquito research. To distinguish transgenic individuals from wild types, genes for fluorescent proteins are the most commonly used genetic markers in transgenic constructs, offering all the advantages of visual selection. Although manual selection under a fluorescence binocular microscope is perfect for the selection of first-generation transgenics, managing established fluorescent lines can be facilitated by complex object parametric analyzer and sorter (COPAS) sorting, which we describe in this protocol. COPAS sorting allows researchers to purify large mosquito larval populations containing only homozygous transgenic individuals, only heterozygotes, or a mix of homozygous, wild types, and heterozygotes in desired proportions. Sorting large populations of a single sex is also possible. Finally, especially when several transgenes of different fluorescence colors are inserted in the same docking site (a recombination site previously inserted in the mosquito genome, which can be used to insert new transgenes into the same locus), they can be maintained together in a single mosquito population to save insectarium space and labor. COPAS sorting can then be used to extract the desired genotype when needed and to readjust transgene frequencies every few generations in case drift is observed.

BackgroundSouth Africa has set a mandate to eliminate local malaria transmission by 2023. In pursuit of this objective a Sterile Insect Technique programme targeting the main vector Anopheles arabiensis is currently under development. Significant progress has been made towards operationalizing the technology. However, one of the main limitations being faced is the absence of an efficient genetic sexing system. This study is an assessment of an An. arabiensis (AY-2) strain carrying the full Y chromosome from Anopheles gambiae, including a transgenic red fluorescent marker, being introgressed into a South African genetic background as a potential tool for a reliable sexing system.MethodsAdult, virgin males from the An. arabiensis AY-2 strain were outcrossed to virgin females from the South African, Kwazulu-Natal An. arabiensis (KWAG strain) over three generations. Anopheles arabiensis AY-2 fluorescent males were sorted as first instar larvae (L1) using the Complex Object Parametric Analyzer and Sorter (COPAS) and later screened as pupae to verify the sex. Life history traits of the novel hybrid KWAG-AY2 strain were compared to the original fluorescent AY-2 strain, the South African wild-type KWAG strain and a standard laboratory An. arabiensis (Dongola reference strain).ResultsThe genetic stability of the sex-linked fluorescent marker and the integrity and high level of sexing efficiency of the system were confirmed. No recombination events in respect to the fluorescent marker were detected over three rounds of introgression crosses. KWAG-AY2 had higher hatch rates and survival of L1 to pupae and L1 to adult than the founding strains. AY-2 showed faster development time of immature stages and larger adult body size, but lower larval survival rates. Adult KWAG males had significantly higher survival rates. There was no significant difference between the strains in fecundity and proportion of males. KWAG-AY2 males performed better than reference strains in flight ability tests.ConclusionThe life history traits of KWAG-AY2, its rearing efficiency under laboratory conditions, the preservation of the sex-linked fluorescence and perfect sexing efficiency after three rounds of introgression crosses, indicate that it has potential for mass rearing. The potential risks and benefits associated to the use of this strain within the Sterile Insect Technique programme in South Africa are discussed.

The distal convoluted tubule (DCT) is instrumental in fine‐tuning of the renal Mg2+ handling. Here, we aim to discover new Mg2+‐related genes in DCT. Transgenic mice expressing eGFP under a DCT‐specific parvalbumin promoter were subjected to Mg2+‐deficient or Mg2+‐enriched diets. Subsequently, the Complex Object Parametric Analyzer and Sorter (COPAS) allowed for the first time isolation of eGFP‐positive DCT cells. RNA extracts thereof were analyzed by DNA microarrays to identify Mg2+ regulatory genes and 46 genes showed differential expression. Several known magnesiotropic genes, such as Trpm6 and Parvalbumin, were upregulated under low dietary Mg2+. Moreover, new genes were identified that are potentially involved in renal Mg2+ handling. Among others, Pcbd1 was identified to be higher expressed in low Mg2+ conditions. Pcbd1 encodes a transcriptional co‐activator of Hnf1b, which was previously implicated in renal Mg2+ handling and maturity onset diabetes of the young (MODY). Interestingly, when examining three independent patients with Pcbd1 mutations, two individuals were diagnosed with hypomagnesemia and renal Mg2+ loss. Moreover, two patients also developed diabetes with characteristics of MODY regardless of the serum Mg2+ levels. By elucidating the Mg2+‐sensitive DCT transcriptome new candidate genes in renal Mg2+ handling have been identified.

Only female mosquitoes consume blood giving them the opportunity to transmit deadly human pathogens. Therefore, it is critical to remove females before conducting releases for genetic biocontrol interventions. Here we describe a robust sex-sorting approach termed SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) that exploits sex-specific alternative splicing of an innocuous reporter to ensure exclusive dominant male-specific expression. Using SEPARATOR, we demonstrate reliable sex selection from early larval and pupal stages in Aedes aegypti, and use a Complex Object Parametric Analyzer and Sorter (COPAS) to demonstrate scalable high-throughput sex-selection of first instar larvae. Additionally, we use this approach to sequence the transcriptomes of early larval males and females and find several genes that are sex-specifically expressed. SEPARATOR can simplify mass production of males for release programs and is designed to be cross-species portable and should be instrumental for genetic biocontrol interventions.

The structural and functional heterogeneity of the collecting duct present a tremendous experimental challenge requiring manual microdissection, which is time-consuming, labor intensive, and not amenable to high throughput. To overcome these limitations, we developed a novel approach combining the use of transgenic mice expressing green fluorescent protein (GFP) in the collecting duct with large-particle-based flow cytometry to isolate pure populations of tubular fragments from the whole collecting duct (CD), or inner medullary (IMCD), outer medullary (OMCD), or connecting segment/cortical collecting duct (CNT/CCD). Kidneys were enzymatically dispersed into tubular fragments and sorted based on tubular length and GFP intensity using large-particle-based flow cytometry or a complex object parametric analyzer and sorter (COPAS). A LIVE/DEAD assay demonstrates that the tubules were >90% viable. Tubules were collected as a function of fluorescent intensity and analyzed by epifluorescence and phase microscopy for count accuracy, GFP positivity, average tubule length, and time required to collect 100 tubules. Similarly, mRNA and protein from sorted tubules were analyzed for expression of tubule segment-specific genes using quantitative real-time RT-PCR and immunoblotting. The purity and yield of sorted tubules were related to sort stringency. Four to six replicates of 100 collecting ducts (9.68+/-0.44-14.5+/-0.66 cm or 9.2+/-0.7 mg tubular protein) were routinely obtained from a single mouse in under 1 h. In conclusion, large-particle-based flow cytometry is fast, reproducible, and generates sufficient amounts of highly pure and viable collecting ducts from single or replicate animals for gene expression and proteomic analysis.