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    About Sony Biotechnology

    • Using cell sorting to isolate cells with genes edited by CRISPR-Cas9

      19 May 2017

      With the ability to easily identify and isolate single cells from a heterogeneous phenotypic population, a cell sorter is a useful tool for laboratories studying and applying the CRISPR technology as a tool for editing genes and creating single cell libraries.

      CRISPR genome editing tools can be used to remove, change or insert DNA sequences into genes. The resulting mutations are maintained in the genome as cells proliferate –in contrast to transient transfection methods.

      Edits to genes are accomplished by directing the Cas9 nuclease to cleave DNA at a specific sequence and a guide RNA (gRNA) into the cell through transfection (or electroporation). For the edit to be completed the cell must be successfully transfected and the DNA cleaved by the Cas9 (referred to as cleavage efficiency).

      Many factors can impact cleavage efficiency including the efficiency of the transfection. A commonly used method to identify cells that have been successfully transfected is to include a plasmid encoding for a fluorescent protein such as eGFP in the transfection along with Cas9 and gRNA.

      Cells can then be sorted based on the expression of the fluorescent protein. The SH800 sorter, lets researchers deposit target cells into 96- or 384 multi-well devices and indexing software records the X and Y well position of each cell. The software can also correlate the fluorescence and scatter phenotype of sorted cells for meta-data analysis.

      The Sony SH800 has been used by several prominent research laboratories to optimize CRISPR expressing cells and to create CRISPR variants with improved function. For more information download this Application Data that demonstrates how the Sony SH800S can be used for single cell sorting of CRISPR/CAS 9 expressing cells.

      Other laboratories have used the SH800 for routine sorting of CRISPR modified cells. Learn more about sorting CRISPR modified cells in the publications below:

      Oakes, Benjamin L., et al. "Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch." Nature biotechnology (2016).

      Sluch, Valentin M., et al. "Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line." Scientific reports 5 (2015).

      Goto, Teppei, et al. "Hypomorphic phenotype of Foxn1 gene-modified rats by CRISPR/Cas9 system." Transgenic research (2016): 1-12.

      Oakes, Benjamin L., Dana C. Nadler, and David F. Savage. "Protein engineering of Cas9 for enhanced function." Methods in enzymology 546 (2014): 491.

      Schrage, Ramona, et al. "The experimental power of FR900359 to study Gq-regulated biological processes." Nature communications 6 (2015).

      Hayashi, Masayasu, et al. "Chd5 Regulates MuERV‐L/MERVL Expression in Mouse Embryonic Stem Cells Via H3K27me3 Modification and Histone H3. 1/H3. 2." Journal of Cellular Biochemistry (2015).


    • How the immune system impacts liver regeneration

      17 May 2017

      Unlike most organs in the human body the liver is able to regenerate allowing donors and patients to re-grow resected tissue. The success and rate of regeneration can be negatively impacted by several factors including sub-septic conditions. One factor is LFA-1, an integrin lymphocyte function-associated antigen-1.

      To better understand how LFA-1 affect liver regeneration, Jörger, et al.1 used a mouse model to determine the functional role of immune cells, especially NKT cells, in response to partial hepatectomy. In this study lymphocytes were characterized on the Sony SP6800 spectral analyzer to determine innate and adaptive immune cells in liver regeneration.  In addition, the study determined that NKT lymphocytes were examined to determine their ability to play a protective role in liver regeneration.

      References

      1. Jörger A-K, Liu L, Fehlner K, Weisser T, Cheng Z, Lu M, et al. (2016) Impact of NKT Cells and LFA-1 on Liver Regeneration under Subseptic Conditions. PLoS ONE 11(12): e0168001. doi.org

    • Immunology 2017 Washington, D.C. May 13-17

      09 May 2017

      Stop by booth #319 at Immunology 2017 for a walk through on how spectral technology, the base of our cell Analyzers, provides better data for research involving heterogeneous samples. You can also get some “face-to-face” time with our newest cell sorter, the FX500 Replaceable Fluidics Cell Sorter. See how the system’s single use consumables support an aseptic workflow and how automation enables walk away operation.

      From May 13 – 17, Immunology 2017 in Washington D.C., is expected to be the largest gathering of immunologists worldwide. The scientific program is packed with presentations from leading immunologists on a full range of topics to stimulate discussion and offer new tools for your research.

      For more information visit Immunology 2017


    • Simple Panels for the Study of T-Cells

      01 May 2017

      T-cells are part of the adaptive immune system with several subtypes including CD8+ effector T-cells and CD4+ helper T-cells. Main categories can be divided into several sub-categories. What are the critical markers to study T-cells? From the key subsets how can other subsets be defined?

      Simplest panel
      This panel identifies the main T-cell populations. These markers are expressed at high levels, as a result there is a lot of choice with fluorochrome selection. Very few cells will be double positive for CD4 and CD8.

      Simplest panel
      Marker Purpose 1 Blue Laser 1 Blue, 1 Red Laser
      CD3 Main marker for the identification of T cells FITC FITC
      CD4 Identification of helper T-cell populations PerCP-Cy5.5 APC
      CD8 Identification of effector T-cell populations PE PE
      CD45 Facilitates gating of leukocytes (optional) PE-Cy7 PerCP-Cy5.5

      When building larger panels these markers can be moved to dimmer fluorochromes. CD3 is sometimes removed from panels to make room for more markers as all CD4+ and CD8+ cells are CD3+.

      Regulatory T-cells (Treg)
      Treg regulate the immune response and are implicated in both cancer and autoimmune disease. In human cells, the most definitive way to identify regulatory T-cells is by utilizing the transcription factor FoxP3. However to access FoxP3 cells must be fixed and permeabilized to access the antigen. Kits and buffer sets, such as True-Nuclear™ Transcription Faction Buffer Set, can simplify experiments and improve results. Once the cells are permeabilized they are not suitable for cell sorting applications. A second Treg panel for sorting is also listed below.

      Human Treg analysis panel with FoxP3

      Human Treg analysis panel with FoxP3
      Marker Purpose 1 Blue Laser 1 Blue, 1 Red Laser
      CD4 Identification of helper T-cell populations PerCP-Cy5.5 FITC
      CD25 Regulatory T-cells express high level of CD25 PE PE
      FoxP3 Identification of regulatory T-cells AlexaFluor® 488 AlexaFluor® 647
      CD45 Facilitates gating of leukocytes (optional) PE-Cy7 PerCP-Cy5.5

      Note: Antibodies against FoxP3 are available and work well conjugated to several fluorochromes. One advantage of the Alexa Fluor dyes is their small size. This can be helpful particularly with intranuclear antigens that are part of a complex.

      Human Treg sorting panel with CD127
      Expression of the IL-7 receptor (CD127) is inversely correlated with FoxP3. CD127 is expressed on the cell surface. Therefore cells do not need to be permeablized and can be sorted. Tregs can be identified as CD4+, CD25+ and CD127-.

      Human Treg sorting panel with CD127
      Marker Purpose 1 Blue Laser 1 Blue, 1 Red Laser
      CD4 Identification of helper T-cell populations PerCP-Cy5.5 FITC
      CD25 Regulatory T-cells express high level of CD25 PE PE
      CD127 Inversely correlated with FoxP3. Can be negatively gated to get Treg cells. AlexaFluor® 488 AlexaFluor® 647
      CD45 Facilitates gating of leukocytes (optional) PE-Cy7 PerCP-Cy5.5

      There are many additional markers available for the characterization of T-cell subsets. The suggested panels provide a starting point for experiments on entry-level instruments. All reagents should be optimized for best results.


    • Explain CRISPR-Cas-9 to your Mom

      01 May 2017

      Thank your super hero mom this Mother’s Day by giving her some quality time and something to remember - the gift of knowing about the work you do that makes the world a better place. To help you with what could be the best little talk ever here’s a 5 minute animation from the Royal Society that explains gene editing and the importance CRISPR-Cas-9 we hope you will find useful. Happy Mother’s Day to all, thank you for what you do.

      YouTube: What is gene editing and how does it work?


    • International Society for Cellular Therapy 2017: Booth 814

      28 Apr 2017

      Please visit us May 3-6, booth 814 for the annual meeting of the International Society for Cellular Therapy (ISCT) in London. The 25th anniversary of program is arranged around cell and gene therapies and tissue engineering applications. Sony will be displaying the FX500 Replaceable Fluidics Cell Sorter. The new system controls sample to sample carryover between experimental runs with single use consumables such as sorting chip, sheath line and sample line to support an aseptic workflow. To streamline workflow, automation guides setup and sort monitoring to enable walk away operation.

      ISCT global association is dedicated to help academic, government and industry sectors transform research into safe and effective cellular therapies to improve human health.


    • Southern California Flow Cytometry’s Flow Summit 2017

      24 Apr 2017

      The SoCal Flow Summit, April 24-25 brings together research, clinical and biomedical scientists at UC Irvine. Scientific presentations include a discussion by Dr. Charles Prussak, Pharm.D, PhD from the Moores Cancer Center at UCSD on recent advances in immune modulating agents targeting PD-1, PDL-1 and CTLA-4 and the potential of cell based therapeutics including those that employ B-, T- and NK-lymphocytes and neutrophils. More on the event at socalflow.org.


    • Cancer Immunotherapy Symposium at UCSD

      17 Apr 2017

      Join us for a poster presentation at the Cancer Immunotherapy Symposium at UCSD Moores Cancer Center on April 20, 2017.  The poster presents data on how the cell sorting workflow can be streamlined with the aid of single use consumables to support a growing range of applications that need controlled conditions. Data presented shows how carryover from sample to sample is minimized by replacing sorting chip, sheath line and sample line to support an aseptic workflow on the FX500 cell sorter from Sony.

      To further support easy workflow, software wizards guide the exchange of these consumables and installation of sheath bags inside a pressurized stainless steel sheath tank. The highly automated system also guides setup and sort monitoring to enable walk away operation.

      Download Poster: Streamlined Workflow for Cell Sorting Using Microfluidics Chip Based Sorter


    • Selecting the Best Clone for Your Flow Cytometry Experiments

      03 Apr 2017

      With literally thousands of commercially available fluorochrome conjugated antibodies for flow cytometry, there are many choices for your application. In many cases several clones are available to the same target. How do you evaluate which clones are best for your research?

      1. Define your application- Are you using live or fixed samples? Will you be looking at intracellular antigens such as cytokines or transcription factors? Some clones perform better than others under different fixation and permeabilization conditions. Evaluate published and manufacturer data for the clone under the conditions in which you plan to use it.
      2. Publications - There are many published flow cytometry panels. One good resource is Optimized Multicolor Immunofluorescence Panels (OMIP) published in Cytometry Part A (http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)15524930/homepage/information_on_omips.htm). OMIPs are optimized, peer-reviewed panels with sufficient information to reproduce the data. These panels provide the specific clone used and detailed information about the sample and other experimental conditions.
      3. HDCM (www.HDCM.org)- runs the Human Leukocyte Differentiation Antigens (HLDA) workshops that characterize and designate CD (cluster of differentiation) markers for human antigens. Started in 1982, this organization sends out new antibodies to volunteer scientists for testing. New antibodies are coded and sent with characterized antibodies for comparison. Results are reviewed and new CD markers and antibodies are designated. The HDCM provides information about which clones have been tested and additional antigen information on their website.
      4. Nonhuman primate cross-reactivity- If you plan to use nonhuman primate samples (baboon, chimpanzee, etc.), the NIH Nonhuman Primate Reagent Resource (http://www.nhpreagents.org/) list clones that have been tested in different nonhuman primate species.
      5. Empirical testing- purchase the available clones and determine which one gives you the best results in your experiment. This is particularly useful if you plan to run the panel several times. Although significant effort has been made to improve the reproducibility of flow cytometry experiments, results can and often vary by laboratory. In house testing of antibodies at several concentrations will provide the best and most consistent results.

      In this article resources from peer reviewed and not for profit organizations have been provided. Determining the right clone for your experiment can improve the quality of your result in addition to saving time and money.


    • FX500 Exchangeable Fluidics Cell Sorter

      03 Apr 2017
      FX500 Internals

      Meeting the need to control sample to sample carryover for sorting applications in academic and industrial settings, such as cell manufacturing, the new FX500 that debuted at the International Society of Stem Cell Research in San Francisco, featuring automation, a fully replaceable fluidics system, and ease of use.

      The novel replaceable fluidics system includes an E-beam irradiated sorting chip and PEEK sample line as well as a gamma irradiated sheath line assembly. The absence of fluidics pumps and inline valves in this design allows for an easy replacement of these single use consumables between experimental runs.

      “The system was designed for real ease of use,” says Allen Poirson, CEO of Sony Biotechnology, “the engineering team built in automation to handle setup, configuration and sort monitoring where technology can very precisely set the system for optimal operation. As for exchanging fluidics, great care was taken to ensure that the fluidics could be replaced by an average person. singlemplifying complex technology is the hallmark of Sony Biotechnlogy systems and this new system, although complex, reflects this goal.”
      FX500 Sheath Tank
      A unique pressurized stainless steel sheath tank encloses a removable rack where sheath bags can be mounted. The sheath fluid from all the bags is channeled via a single sheath line to the chip. The E beam sterilized sorting chip acts as an integrated flow cell-nozzle and can be easily installed and removed. The sample and sheath fluid are injected into the chip's micro-fluidic channels via inlet ports. Excitation lasers interrogate the sample within the chip before it passes through and is sorted as droplets. The chip is directly connected to the waste line and can be declogged via software options.

      Unlike traditional systems that require nozzles to be removed for manual cleaning, the FX500 system uses an automated purge and prime cycle triggered by the software to clear the chip. This allows for continuous use without removing the chip. The chip is available in 70μm, 100μm and 130μm sizes allowing researchers to easily adapt the system to cell sizes.

      The FX500 can be configured with up to three lasers, and offers sort deposition system to handle multi-well plates and a custom biosafety cabinet as options. More info on the system