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    • Similar but not the same - Genome sequencing of sorted single cells

      09 Nov 2017

      Gene expression profiling is a powerful approach that promotes a deeper understanding of the characteristics of cells at the transcriptional level and has broad implications for basic and clinical research. Historically, gene expression profiling has been performed on populations of cells, typically cell lysates, where observed expression levels represent an average of the unique expression states of each cell within the population. Such phenotypically similar populations can be purified in bulk by flow cytometry based cell sorting prior to RNA analysis. 1,2

      There are several challenges with bulk analysis. For example, these approaches may not detect subtle, but potentially biologically meaningful differences between seemingly identical cells. It is now widely recognized that within a population, cells dramatically vary with respect to behavior during their lifespan and this variation is reflected in their transcriptome expression levels. The recent emergence of single cell RNA analysis (RNA- seq) has provided an important means to discern and profile cell-to-cell variability on a genomic scale. The isolation of single cells required for construction of genomic libraries can be performed using flow cytometry based cell sorting.

      The Sony SH800 cell sorter has several features that support the isolation of single cells for RNA-seq. The Sony SH800 is an automated system that is used for efficiently sorting individual cells in a high throughput manner using 96 or 384 well formats with high precision. This system operates on different microfluidics sorting chips with orifice of 70, 100 and 130um enabling single cell sorting of a wide range of cell sizes from small nuclei3 and immune cells4) to large cylindrically shaped iPSC derived cardiomycocytes5 in addition to cryopreserved tissue derived from mouse disease models6. RNA-seq is a very sensitive application and therefore, care must be taken to avoid creating experimental artifacts while isolating cells. To achieve high sensitivity within a dynamic range of RNA expression, single cells isolated for RNA-seq must have low damage and intact RNA. The SH800 can be operated under low sheath and sample pressure to reduce shear stress on cells. Additionally, the sorting chip can be exchanged between samples to control for the presence of nucleases such as RNase enabling optimal sample integrity for downstream gene expression analysis.

      The phenotypic data of individual cells sorted using SH800 can be indexed using software options so that it can be bioinformatically integrated with gene expression assays to produce a molecular dataset of each cell and linked to its functional activity.

      References

      1. Genome-wide identification of inter-individually variable DNA methylation sites improves the efficacy of epigenetic association studies. Hachiya T, Furukawa R, Shiwa Y, Ohmomo H, Ono K, Katsuoka F, Nagasaki M, Yasuda J, Fuse N, Kinoshita K, Yamamoto M, Tanno K, Satoh M, Endo R, Sasaki M, Sakata K, Kobayashi S, Ogasawara K, Hitomi J, Sobue K and Shimizu A.
      2. Protein engineering of Cas9 for enhanced function. Oakes BL, Nadler DC, and Savage DF. Methods Enzymol. 2014; 546: 491–511.
      3. Sequencing thousands of single-cell genomes with combinatorial indexing. Nat Methods 2017, 14(3): 302-308. Vitak SA, Torkenczy KA, Rosenkrantz JL, Fields AJ, Christiansen L, Wong MH, Carbone L, Steemers FJ, and Adey A.
      4. Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells. Minami H, Tashiro K, Okada A, Hirata N, Yamaguchi T, Takayama K, Mizuguchi H, Kawabata K.  PLoS ONE 10(6): e0128890. doi:10.1371/journal.pone.0128890
      5. Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy. Cell Reports 2017, 18: 571–582. Cho GS, Lee DI, Tampakakis E, Murphy S, Andersen P, Uosaki H, Chelko S, Chakir K, Hong I, Seo K, Chen HV, Chen X, Basso C, Houser SR, Tomaselli GF, O’Rourke B, Judge DP, Kass DA, and Kwon C.
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    • Detection of in vivo cell movement by flow cytometry

      16 Oct 2017

      Much of cell research happens in the context of cultured or modified cells. More biologically relevant experiments can be done with blood. However, blood cannot entirely capture what takes place within a live organism. In their paper Futamura, et al.1 describes a method to study the movement and subsequent interactions of immune cells in live mice by flow cytometry.

      To study cell movement, KikGR knock-in mice were used.2 KikGR mice express the photoconvertible fluorescent protein KikGR in a cell-specific manner. KikGR fluorescence changes irreversibly from green to red upon exposure to violet light. In these experiments, inguinal lymph node (LN) of KikGR mice were exposed to violet light for photo-conversion of KikGR.  All of the cells in the inguinal LN were subsequently labeled with KikGR-Red signal. Twenty-four hours after photo-conversion, the cell migration from the photo-converted LN to other anatomical organs and the subsequent replenishment of cells in photo-converted LN was studied with an 11-color panel using an SP6800 spectral cytometer.

      With the SP6800, phenotyping the T and B cell subsets of the LN was easily obtained with spectral unmixing without the usual complexities of color compensation.  Furthermore, cellular autofluorescence of the various cell types assisted in phenotyping the subsets. Read more about this research at http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22725/full.

       

      References:

      1 Futamura, Koji, et al.  Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.   Cytometry Part A 2015:87:9  pp. 830-842 doi:10.1002/cyto.a.22725  http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22725/full

      2 Tomura M, Hata A, Matsuoka S, et al. Tracking and quantification of dendritic cell migration and antigen trafficking between the skin and lymph nodes. Scientific Reports. 2014;4:6030. doi:10.1038/srep06030. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4129424/

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    • Obtaining single cell suspensions of brain tissues

      29 Sep 2017

      Recent advances in genome editing and the application of fluorescent proteins have accelerated Interest in isolating specific populations of brain cells from mixed populations. Researchers are also using cell sorting to isolate single cells for expansion and analysis.

      Follow these 5 guidelines to address the challenge of obtaining single cell suspensions from delicate brain tissues.

      1. The methods chosen to dissociate brain tissue for obtaining single cells should be gentle and impart low shear stress in order to preserve cell surface antigens and cell viability.
      2. The desired region of the brain is dissected and the relevant tissue is minced.
      3. Cells are further isolated through enzymatic digestion using papain or Accutase with EDTA.
      4. Large debris and cell clusters should be removed by filtering through mesh prior to staining and sorting.
      5. Viability dyes such as DAPI should be included to monitor cell death. The number of dead cells can be used to determine consistency and quality of cell preparation.

      Studies

      Several studies have been published in which scientists have successfully isolated stem cells from whole brain homogenate using cell sorting. The references below provide more detailed information about preparing and sorting brain tissue.

      1. Daynac, Mathieu, Lise Morizur, Thierry Kortulewski, Laurent R. Gauthier, Martial Ruat, Marc-André Mouthon, and François D. Boussin. "Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics." Journal of Visualized Experiments: JoVE (103 (2015). https://www.ncbi.nlm.nih.gov
      2. Wylot, Bartosz, et al. "Isolation of vascular endothelial cells from intact and injured murine brain cortex—technical issues and pitfalls in FACS analysis of the nervous tissue."Cytometry Part A 10 (2015): 908-920. http://onlinelibrary.wiley.com
      3. Guez-Barber, Danielle, et al. "FACS purification of immunolabeled cell types from adult rat brain."Journal of neuroscience methods 1 (2012): 10-18. https://www.ncbi.nlm.nih.gov
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    • Gaining an edge – taking advantage of novel fluorescent proteins with Flow Cytometry

      18 Sep 2017

      Placing several fluorescent proteins together in a flow cytometry panel offers greater power and capability for experiments. However, handling autofluorescent signal with fluorescent proteins is out of reach for conventional flow cytometetry users. Sony spectral flow cytometry analyzers enable researchers to harness up to five near infra-red fluorescent proteins in a single experiment. Moreover, spectral technology lets users accurately identify autofluorescence, and eliminate it if needed.

      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.

      Flow cytometry is a popular and common method for the detection of fluorescent proteins. However, conventional flow cytometers rely on optical filters to separate overlapping signals, which does not allow some combinations of fluorescent proteins to be resolved or compensated correctly. In contrast, spectral flow cytometry captures has no optical filters and uses 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 infared (NIR) lasers. Conventional flow cytometry can only detect two of the five iRFP at the same time. Spectral flow cytometry can detect all five fluorescent proteins at once. Combining these new fluorescent proteins with spectral technology will expand the number of fluorescent 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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    • 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

      Twitter

      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

      Bacteria such as E. coli are popular model systems for engineering and production of modified proteins. Yet the idea of putting bacteria in a cell sorter conjures unwelcome images. Take heart, the Sony SH800 cell sorter, simplifies decontamination by quickly and easily letting researchers replace key components that come in contact with the sample. Here are some examples, and publication citations.

      Here are some examples of publications and applications where transfected bacteria are detected and sorted on the basis of either permeant fluorescent dyes or by the fluorescent proteins that they express using  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. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641071/). This 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 6 (2016): 646-651. (http://europepmc.org/articles/pmc4900928)
      1. Engineering of proteins for enhanced production. 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.
      1. 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 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.
      1. 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.

      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 Sony 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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    • 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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