Project description:The male karyotypes of Acmaeodera pilosellae persica Mannerheim, 1837 with 2n=20 (18+neoXY), Sphenoptera scovitzii Faldermann, 1835 (2n=38-46), Dicerca aenea validiuscula Semenov, 1895 - 2n=20 (18+Xyp) and Sphaerobothris aghababiani Volkovitsh et Kalashian, 1998 - 2n=16 (14+Xyp) were studied using conventional staining and different chromosome banding techniques: C-banding, AgNOR-banding, as well as fluorochrome Chromomycin A3 (CMA3) and DAPI. It is shown that C-positive segments are weakly visible in all four species which indicates a small amount of constitutive heterochromatin (CH). There were no signals after DAPI staining and some positive signals were discovered using CMA3 staining demonstrating absence of AT-rich DNA and presence of GC-rich clusters of CH. Nucleolus organizing regions (NORs) were revealed using Ag-NOR technique; argentophilic material mostly coincides with positive signals obtained using CMA3 staining.
Project description:The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. Whole mount preparations preserve the structure of meiotic nuclei, and importantly, each gonad arm contains all stages of meiosis, organized in a temporal-spatial progression that makes it easy to identify nuclei at different stages. Adult hermaphrodites have two gonad arms, each organized as a closed tube with proliferating germline stem cells at the distal closed end and cellularized oocytes at the proximal open end, which join in the center at the uterus. Dissection releases one or both gonad arms from the body cavity, allowing the entirety of meiosis to be visualized. Here, a common protocol for immunofluorescence against a protein of interest is presented, followed by DAPI staining to mark all chromosomes. Young adults are immobilized in levamisole and quickly dissected using two syringe needles. After germline extrusion, the sample is fixed before undergoing a freeze crack in liquid nitrogen, which helps permeabilize the cuticle and other tissues. The sample can then be dehydrated in ethanol, rehydrated, and incubated with primary and secondary antibodies. DAPI is added to the sample in the mounting medium, which allows reliable visualization of DNA and makes it easy to find animals to image under a fluorescent microscope. This technique is readily adopted by those familiar with handling C. elegans after a few hours spent practicing the dissection method itself. This protocol has been taught to high-schoolers and undergraduates working in a research lab and incorporated into a course-based undergraduate research experience at a liberal arts college.
Project description:Though representing a major component of eukaryotic biodiversity, many microbial eukaryotes remain poorly studied, including the focus of the present work, testate amoebae of the order Arcellinida (Amoebozoa) and non-model lineages of ciliates (Alveolata). In particular, knowledge of genome structures and changes in genome content over the often-complex life cycles of these lineages remains enigmatic. However, the limited available knowledge suggests that microbial eukaryotes have the potential to challenge our textbook views on eukaryotic genomes and genome evolution. In this study, we developed protocols for DAPI (4',6-diamidino-2-phenylindole) staining of Arcellinida nuclei and adapted protocols for ciliates. In addition, image analysis software was used to estimate the DNA content in the nuclei of Arcellinida and ciliates, and the measurements of target organisms were compared to those of well-known model organisms.The results demonstrate that the methods we have developed for nuclear staining in these lineages are effective and can be applied to other microbial eukaryotic groups by adjusting certain stages in the protocols.
Project description:Malassezia is a lipophilic commensal yeast that resides mainly on the mammalian skin and is also found to associate with the internal organs. Dysbiosis of Malassezia is related to several diseases and often escapes detection as it is difficult to culture and maintain. Malassezia cell wall differs from other budding yeasts like S. cerevisiae due to the difference in the lipid content and is difficult to transform. In this study, we present a methodology to stain Malassezia's nucleus and perform cell cycle studies. However, staining presents a challenge due to its exceptionally thick cell wall with high lipid content, hindering conventional methods. Our novel methodology addresses this challenge and enables the staining of the Malassezia nucleus with a low background. This would allow researchers to visualize the overall nuclear health specifically nuclear morphology and analyze DNA content, crucial for cell cycle progression. By employing DNA-specific dyes like DAPI or Hoechst, we can observe the nuclear structure, and using PI we can differentiate cells in distinct cell cycle phases using techniques like flow cytometry. This novel staining methodology unlocks the door for in-depth cell cycle analysis in Malassezia which has challenged us through ages being refractory to genetic manipulations, paving the way for a deeper understanding of this commensal fungus and its potential role in human health.
Project description:Screening for bacteria with abilities to accumulate valuable intracellular compounds from an environmental community is difficult and requires strategic methods. Combining the experimental procedure for phenotyping living cells in a microbial community with the cell recovery necessary for further cultivation will allow for an efficient initial screening process. In this study, we developed a strategy for the isolation of polyphosphate-accumulating organisms (PAOs) by combining (i) nontoxic fluorescence staining of polyphosphate granules in viable microbial cells and (ii) fluorescence-activated cell sorting (FACS) for the rapid detection and collection of target cells. To implement this screening approach, cells from wastewater sludge samples were stained with 4'6-diamidino-2-phenylindole (DAPI) to target cells with high polyphosphate (polyP) accumulation. We found a staining procedure (10 μg/ml of DAPI for 30 min) that can visualize polyP granules while maintaining viability for the majority of the cells (>60%). The polyP positive cells were recovered by FACS, purified by colony isolation and phylogenetically identified by 16S rRNA gene sequencing. Follow-up analysis confirmed that these isolates accumulate polyP, indicating that DAPI can be implemented in staining living cells and FACS can effectively and rapidly screen and isolate individual cells from a complex microbial community.
Project description:Recent studies taking advantage of automated single-cell time-lapse analysis have reignited interest in the bacterial cell cycle. Several studies have highlighted alternative models, such as Sizer and Adder, which differ essentially in relation to whether cells can measure their present size or their amount of growth since birth. Most of the recent work has been done with Escherichia coli. We set out to study the well-characterized Gram-positive bacterium, Bacillus subtilis, at the single-cell level, using an accurate fluorescent marker for division as well as a marker for completion of chromosome replication. Our results are consistent with the Adder model in both fast and slow growth conditions tested, and with Sizer but only at the slower growth rate. We also find that cell size variation arises not only from the expected variation in size at division but also that division site offset from mid-cell contributes to a significant degree. Finally, although traditional cell cycle models imply a strong connection between the termination of a round of replication and subsequent division, we find that at the single-cell level these events are largely disconnected.
Project description:Microchips are fundamental tools for single-cell analysis. Although various microfluidic methods have been developed for single-cell trapping and analysis, most microchips cannot trap single cells deterministically for further analysis. In this paper, we describe a novel resistance-based microfluidic chip to implement deterministic single-cell trapping followed by immunofluorescence staining based on the least flow resistance principle. The design of a large circular structure before the constriction and the serpentine structure of the main channel made the flow resistance of the main channel higher than that of the trapping channel. Since cells preferred to follow paths with lower flow resistance, this design directed cells into the capture sites and improved single-cell trapping efficiency. We optimized the geometric parameters using numerical simulations. Experiments using A549 and K562 cell lines demonstrated the capability of our chip with (82.7 ± 2.4)% and (84 ± 3.3)% single-cell trapping efficiency, respectively. In addition, cells were immobilized at capture sites by applying the pulling forces at the outlet, which reduced the cell movement and loss and facilitated tracking of the cell in real time during the multistep immunofluorescence staining procedure. Due to the simple operation, high-efficiency single-cell trapping and lower cell loss, the proposed chip is expected to be a potential analytical platform for single tumor cell heterogeneity studies and clinical diagnosis.
Project description:Herein proposed is a simple system to realize hands-free labeling and simultaneous detection of two human cell lines within a microfluidic device. This system was realized by novel covalent immobilization of pH-responsive poly(methacrylic acid) microgels onto the inner glass surface of an assembled polydimethylsiloxane/glass microfluidic channel. Afterwards, selected thiophene labeled monoclonal antibodies, specific for recognition of CD4 antigens on T helper/inducer cells and CD19 antigens on B lymphocytes cell lines, were encapsulated in their active state by the immobilized microgels. When the lymphocytes suspension, containing the two target subpopulations, was flowed through the microchannel, the physiological pH of the cellular suspension induced the release of the labeled antibodies from the microgels and thus the selective cellular staining. The selective pH-triggered staining of the CD4- and CD19-positive cells was investigated in this preliminary experimental study by laser scanning confocal microscopy. This approach represents an interesting and versatile tool to realize cellular staining in a defined module of lab-on-a-chip devices for subsequent detection and counting.
Project description:Alpha-herpesviruses, including herpes simplex virus and pseudorabies virus (PRV), infect the peripheral nervous system (PNS) of their hosts. Here, we describe an in vitro method for studying neuron-to-cell spread of infection as well as viral transport in axons. The method centers on a novel microfluidic chamber system that directs growth of axons into a fluidically isolated environment. The system uses substantially smaller amounts of virus inoculum and media than previous chamber systems and yet offers the flexibility of applying multiple virology and cell biology assays including live-cell optical imaging. Using PRV infection of cultured PNS neurons, we demonstrate that the microfluidic chamber recapitulates all known facets of neuron-to-cell spread demonstrated in animals and other compartmented cell systems.
Project description:Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy.