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    • What do olives and cell sorting have in common?

      23 Aug 2017

      Bacteria, most notably the gut microbiome, have gained increased attention for their role in agriculture and human disease. However study of individual bacterium can be challenging since most bacteria do not grow under simple culture conditions. In a recent study, Blow1, et al. have published a draft sequence of the bacteria, Candidatus Erwinia dacicola (Enterobacteriaceae). This bacteria has a symbiotic relationship with the Bactrocera oleae, also known as the olive fly. The larva of the olive fly grow in unripened olives leading to reduced crop yields. The larva has adapted to the special environment of the unripened olive with help from Candidatus Erwinia dacicola bacteria. Without bacteria the larva do not develop.

      A key challenge for the characterization of Candidatus Erwinia dacicola is the inability to culture this bacteria. It must be isolated from natural sources which are contaminated with other cells. Therefore, to isolate this bacteria researchers isolated cells from the guts of olive flies. Cells were stained with CellTracker and then sorted on a Sony SH800. Genomic DNA was amplified and used to construct a library which was then used for sequencing.

      Understanding this bacteria may lead to improved control of the olive fly and increased yields of olives.

       

      1 Blow, Frances, et al. "Draft Genome Sequence of the Bactrocera oleae Symbiont “Candidatus Erwinia dacicola”." Genome Announcements 4.5 (2016): e00896-16.

      http://genomea.asm.org/content/4/5/e00896-16.full

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      A study offers a draft sequence of a Candidatus Erwinia dacicola bacteria isolated from olive fly gut, which reduces olive crop yield.

      See how a study offers a draft sequence of a Candidatus Erwinia dacicola bacteria isolated from olive fly gut.

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    • Five ways to save money on antibodies for flow cytometry

      11 Aug 2017
      1. Purchase larger sizes of common antibodies on established fluorochromes. Packaging and shipping antibodies can be costly. Therefore smaller sizes typically cost greater than two times more compared to larger sizes.
      2. Titrate your antibodies for optimal performance in your assay. In the majority of cases you will use a lot less antibody and have better results.
      3. Properly store your antibodies. Most fluorochrome labeled antibodies contain a preservative such as sodium azide and are best stored at 4°C protected from light. Fluorochrome labeled antibodies can have shelf lives of years if they are stored properly. If you purchase a larger size that you intend to use over an extended period consider aliquoting into amber tubes. This will minimize exposure to light and room temperature increasing shelf life.
      4. Use low retention pipet tips and tubes. Antibodies and other proteins can nonspecifically stick to tubes and pipette tips. This can not only result in the need to use more antibody, but also inconsistent results.
      5. Mix conjugated antibodies prior to use. Antibodies can settle over time. If they are not properly mixed before use, insufficient quantities will be present in your experiment. This settling can also lead to inconsistent staining from day to day.
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    • Resources for flow cytometry standardization

      09 Aug 2017

      Numerous consortia today are working to achieve standardized approaches to important topics for research and clinical applications. The work they do to achieve reproducible results is critical to good research results and reliable clinical diagnosis and outcomes.  In this series, we will highlight standardization efforts from The Human Immune Phenotyping Consortium (HIPC), the EuroFlow Consortium, and the ONE Study. These studies contain detailed panels and strategies to improve the reproducibility of flow cytometry.

      The Human Immune Phenotyping Consortium

      Established in 2010, The Human Immune Phenotyping Consortium (HIPC) (https://www.immuneprofiling.org) was developed by the Federation of Clinical Immunology Societies (FOCIS) to address standardization across flow cytometry assays.

      This program has expanded to provide centralized research resources for the comprehensive understanding of the human immune system.

      In their 2016 Nature publication, the HIPC describes their standardization study. Identified sources of experimental variability include: combinations of markers and fluorochromes, sample handling, instrument type and set up, gating and analysis strategies, and ways in which data are reported.

      To control for markers and fluorochromes, the HIPC immunophenotyping experts developed five standardized panels as pre-configured, lyophilized reagents in 96-well plates consisting of eight-color antibody cocktails to phenotype major immune cell subsets in peripheral blood mononuclear cells (PBMC). The plates along with lyophilized control PBMCs and a consensus detailed staining protocol were distributed to nine international laboratories.

      Data collected from this study were analyzed manually at each site. FCS files were sent to a central site where they were analyzed using both manual and automated gating. Central manual gating was found to significantly reduce the variability across datasets. Larger more easily identified subsets such as CD3+, CD4+ T-cells had less variability compared to smaller, dimmer subsets. In addition, subsets that required multiple successive gates had higher CVs. Automated gating algorithms gave similar results to central gating except in the cases of rare subsets and poorly resolved populations. In some cases, these differences could be attributed to subtle differences in the manual gating of upstream subsets.

      This paper also contains detailed panels and a table describing which populations of cells could be reliably detected by automated gating in their study. Information provided may help laboratories to set up their own SOPs especially for the detection of rare and dim subsets.

      Finak, Greg, et al. "Standardizing flow cytometry immunophenotyping analysis from the human immunophenotyping consortium." Scientific reports 6 (2016). www.nature.com/

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    • Four reasons to put bacteria into your cell sorter

      27 Jul 2017

      Four reasons to put bacteria into your cell sorter

      For many of us the idea of putting bacteria in a cell sorter conjures images of cell culture contamination, the smell of bleach, and hours of cleaning. With the SH800 cell sorter, that is not the case because decontamination is simplified by quickly replacing the key components of the cell sorter that come in contact with the sample such as the sample line and sorting chip.  Bacteria such as E. coli are popular model systems for engineering and production of modified proteins. Such transfected bacteria are detected and sorted on the basis of either permeant fluorescent dyes or by the fluorescent proteins that they express.

      Here are some examples of publications and applications where the SH800 has been used to sort bacteria:

      1. CRISPR/Cas9 optimization. The CRISPR/Cas9 system has opened new possibilities in the treatment of disease. However, for the CRISPR system to reach its full clinical utility better understanding of targeting and improved function are critical. Scientists at the University of California, Berkley have transformed bacteria with constructs containing GFP, and sorted cells to identify insertion hot spots to engineer Cas9 for enhanced function.
        • Oakes, Benjamin L. et. al. "Protein engineering of Cas9 for enhanced function."Methods in enzymology 546 (2014): 491. www.ncbi.nlm.nih.gov
        • The paper details methods for screening engineered Cas9 protein. The strategy described may be useful for the screening of other engineered proteins.
        • Oakes, Benjamin L., et al. "Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch." Nature biotechnology 34.6 (2016): 646-651. http://europepmc.org/
      2. Engineering of proteins for enhanced production. E. Coli are routinely used for protein production but they can also be used for the production of other molecules such as polyester. Many bacteria, including E. coli produce polymer inclusions, which serve as stockpiled carbon storage material during periods of nutrient imbalance. These polymer inclusions can be leveraged to make polyesters that are an alternative to petroleum based-plastics. To increase the yield of these polyesters efforts have been made to engineer the bacteria. The SH800 was used in these studies to study the cells density of the bacteria.
        • Kadoya, Ryosuke, et al. "MtgA deletion-triggered cell enlargement of Escherichia coli for enhanced intracellular polyester accumulation." PLOS one 10.6 (2015): e0125163. http://journals.plos.org/
        • Kadoya, Ryosuke, et al. "Genome-wide screening of transcription factor deletion targets in Escherichia coli for enhanced production of lactate-based polyesters."Journal of Bioscience and Bioengineering (2017). http://www.sciencedirect.com/
      3. Development of new expression systems. Inducible expression systems can turn protein production on or off to varying degrees by treating the culture with a drug or other molecule. They are particularly useful for the expression proteins that may be toxic to the cell when expressed at high levels. Most available inducible expression systems are known to be “leaky” meaning that there may still be some expression even when expression is “off”. Researchers at the University of Manchester have created a systems that contains both an inducible promoter and an orthogonal riboswitch to control gene expression in several strains of E. coli. The constructs contained both the inducible elements and eGFP (green fluorescent protein). The SH800 was used to identify subpopulations of bacteria with differing levels of GFP expression.
        • Morra, Rosa, et al. "Dual transcriptional-translational cascade permits cellular level tuneable expression control."Nucleic acids research (2015): gkv912. https://academic.oup.com/
        • This paper also contains a nice introduction on the principles of inducible expression systems.
      4. Identification and enumeration of hard to culture bacteria. While many bacteria such as coli are relatively easy to grow in culture, some such as those found in the gut microbiome are not. Cell sorting is a useful way to enumerate bacteria from natural sources that contain mixtures of cells. To better understand the role of the bacteria Candidatus Erwinia dacicola in the life cycle of the olive fly, it was isolated by sorting cells from the gut of the fly and sequenced.
        • Blow, Frances, et al. "Draft Genome Sequence of the Bactrocera oleae Symbiont “Candidatus Erwinia dacicola”."Genome Announcements 5 (2016): e00896-16. http://genomea.asm.org/

       

      Sorting of bacteria is a useful tool for protein engineering and the study of bacteria. The studies cited above are practical examples of how you can incorporate the sorting of bacteria into your standard laboratory practices. But don’t worry, post bacteria sort your SH800 will be easy to clean and set up for your next mammalian cell experiment.

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    • Learning more about CRIPSR from the experts – Dr. Jennifer Doudna

      25 Jul 2017

      Listen to Dr. Jennifer Doudna, one of the discoverers of CRISPER systems discusses how the new genome engineering technology was discovered.

      Video: Genome Engineering with CRISPR-Cas9: Birth of a Breakthrough Technology

      As part of her work understanding how RNA molecules control gene expression in bacteria and eukaryotic cells, Dr. Doudna’s laboratory began to study to mechanism of CRISPR, a part of the bacterial genomic immune system. In 2011 Doudna met Dr. Emmanuelle Charpentier (who currently holds several titles including Director at the Max Plank Institute for Infection Biology) at a scientific conference. After this meeting both labs collaborated to determine the function of Cas9. This important work led to the discovery that Cas9 could specifically cut DNA at desired sequences. Today the Doudna lab explores the mechanistic understanding of fundamental biological process involving RNA

      Dr. Doudna is a member of the departments of Molecular and Cell Biology at UC Berkely, the Howard Hughes Medical Institute, and Lawrence Berkeley National Lab, along with the National Academy of Sciences, and the American Academy of Arts and Sciences.  She has been lauded for her contributions to the field of biochemistry, with numerous prestigious awards and fellowships. In 2017 she received the Japan Prize for original and outstanding achievements in science and technology (jointly with Emmanuelle Charpentier) and the F. Albert Cotton Medal for excellence in chemical research.

      You can also see Dr. Doudna talk about the ethical challenges of CRISPR at her TED Talk.

      Video: How CRISPR lets us edit our DNA

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    • Spectral flow cytometry allows for the use of novel fluorescent proteins

      11 Jul 2017

      In 2008 the Nobel Prize in Chemistry was given to Shimomura, Chalfie and Tsien for the discovery and development of green fluorescent protein (GFP). This endogenously fluorescent protein was originally cloned from the jellyfish Aequorea Victoria.1 Since that time it has been extensively modified to improve performance in mammalian systems and to expand the palette of fluorescent proteins. This has resulted in new fluorescent proteins that are excited and detected at different wavelengths, allowing more proteins to be detected simultaneously.

      One limitation for the development and use of new fluorescent proteins is the ability to detect them. Flow cytometry is a popular method for the detection of fluorescent proteins. However, with conventional flow cytometers that rely on optical filters to separate overlapping signals, some combinations of fluorescent proteins cannot be resolved. In contrast, spectral flow cytometry captures all signals and relies on spectral unmixing to separate highly overlapping fluorochromes.

      To expand the number of available fluorescent proteins, Telford, et al.2 examined members of the iRFP series, proteins isolated from bacterial phytochromes excited by red and near infrared (NIR) lasers. Using conventional flow cytometry only two could be detected at the same time. With spectral flow cytometry all five fluorescent proteins could be used together. Combining these new fluorochromes with spectral technology will expand the number of proteins that can be studied together in the same experiment.

      1Tsien, Roger Y. et. al.  “The Green Fluorescent Protein” Annual Review of Biochemistry 67 (1998): 509-544. doi: 10.1146/annurev.biochem.67.1.509

      2Telford WG et. al. “Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes.” PLoS ONE 10(3) (2015): e0122342. doi:10.1371/journal.pone.0122342

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    • Will small amounts of preservative kill my cells?

      11 Jul 2017

      Most commercially available antibodies contain small amounts of preservatives such as sodium azide to prevent microbial growth. However, sodium azide is also toxic to mammalian cells as it inhibits cellular respiration. Actual toxicity varies by cell type with neuronal cells being most sensitive.1 Toxicity is concentration, time, and temperature dependent. For most cell sorting experiments the health of cells are not impacted because the antibody is diluted and cells are typically incubated on ice for less than one hour.

      For experiments where cells will be incubated with antibodies over several hours or days, sodium azide free formulations are recommended. Antibodies can be purchased in this formulation or sodium azide can be removed by dialysis. However it is important to note that these antibodies will be significantly more susceptible to microbial contamination. If they are purified they can be aliquoted and frozen.

       

      1Ishikawa, Takaki, Bao-Li Zhu, and Hitoshi Maeda. "Effect of sodium azide on the metabolic activity of cultured fetal cells." Toxicology and industrial health22.8 (2006): 337-341.

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    • Learn About Spectral Flow and DeNovo FCS Express 6

      06 Jul 2017

      Join us for a free webinar on Spectral Flow and FCS Express 6 which provides native support for Sony spectral data files. See how spectral flow cytometry delivers better data and simplifies panel design. In addition we’ll show how seamless integration between FCS Express and Sony spectral flow cytometry analyzers allows you to move quickly from acquisition to expanded data visualization with spectral overlays, tSNE, Spade, and plate based heat maps.

      Register: Spectral Flow Cytometry and Data Analysis Techniques Webinar

      Data Sheet: De Novo FCS Express Software 6.0 for SA3800 and SP6800 Spectral Analyzers

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    • Enabling flow cytometry analysis of tissues with spectral flow cytometry

      23 Jun 2017

      Flow cytometry has long been considered a tool of hematologists and immunologists who primarily work with the hematopoetic system.  Flow cytometry is capable of performing the simultaneous detection of more than 20 parameters; however it requires the sample to be prepared into a single cell suspension. The need for a single cell suspension makes the sample preparation more intensive for cells derived from tissues.  Many tissues are also highly autofluorescent, which results in background noise that limits detection of low levels of expression on conventional flow cytometers.

      Researchers from the Institut Pasteur, Immunology Department in Paris France have published a study in PLoS One1 which simultaneously measured 21 parameters (19 fluorochromes) on cells derived from mouse embryonic heart and intestine. Both tissue types contain autofluorescent cells. With conventional cytometers, specific band pass filters must be carefully selected to enable compensation for spectral overlap.  The more overlap between fluorescent dyes, the more difficult to perform conventional compensation. Furthermore, cells such as those described in the article have larger amounts of inherent autofluorescence complicating the data analysis. With the SP6800 a spectral unmixing algorithm is used to determine the abundance of signal from each fluorochrome. This allows for the separation of highly overlapping signals including GFP and FITC as well as effective management of autofluorescence. Directly comparing results on two conventional flow cytometers and the SP6800, only the SP6800 was capable of effective phenotypic resolution of the target populations.

      REFERENCES

      1 Schmutz S, Valente M, Cumano A, Novault S (2016) Spectral Cytometry Has Unique Properties Allowing Multicolor Analysis of Cell Suspensions Isolated from Solid Tissues. PLoS ONE 11(8): e0159961. journals.plos.org

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    • Learning more about CRISPR from experts

      23 Jun 2017

      Listen to Dr. Feng Zhang who was first to adapt CRISPR-Cas9 for genome editing on eukaryotic cells explain CRISPR in this 2 minute video: Questions And Answers About CRISPR

      Today Dr. Zhang leverages CRISPR and other methodologies to study the role of genetic and epigenetic mechanisms underlying diseases, specifically focusing on disorders of the nervous system. He is especially interested in complex disorders, such as psychiatric and neurological diseases, that are caused by multiple genetic and environmental risk factors and which are difficult to model using conventional methods. His lab is focused on developing novel tools to better understand and treat neuropsychiatric diseases and applying these novel tools to interrogate gene function in animal and stem cell models.  Zhang’s methods are also being used in the fields of immunology, clinical medicine, cancer biology, and other areas of research. Zhang’s long-term goal is to develop novel therapeutic strategies for disease treatment.

      Dr. Zhang is the James and Patricia Poitras Professor in Neuroscience at the McGovern Institute for Brain Research and in the departments of Brain and Cognitive Sciences and Biological Engineering at the Massachusetts Institute of Technology (MIT). He also has appointment with the Broad Institute of MIT and Harvard.

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