![References
Abcam.com 2016, Direct vs indirect immunofluorescence | Abcam., online, viewed 24 September 2016, <http://www.abcam.com/secondary-
antibodies/direct-vs-indirect-immunofluorescence>.
Bio.davidson.edu 2001, FACS Methodology, viewed 21 September 2016, <http://www.bio.davidson.edu/courses/genomics/method/facs.html>.
Blogs.sun.ac.za n.d., BD FACS Calibur », viewed 21 September 2016, <http://blogs.sun.ac.za/fmc/bd-facs-calibur/>.
Bronte, V. and Gabrilovich, D. 2016, Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.
Nature Communications, 7, p.12150.
Catherine, M., Brian T, F. and Timothy C, F. 2010, Fluorescence-Activated Cell Sorting for CGMP Processing of Therapeutic Cells. 1st ed.
[ebook] Sparks: BD biosciences, p.7, viewed 23 September 2016,
<https://www.researchgate.net/profile/Timothy_Fong2/publication/228470167_FluorescenceActivated_Cell_Sorting_for_CGMP_Processing_of
_Therapeutic_Cells/links/55f6ef6e08ae07629dbb159e.pdf>.
Dartmouth Undergraduate Journal of Science 2008, 488 nm: A Review of FACS Technology and its Application in Biological Research, online,
viewed 23 September 2016, < http://dujs.dartmouth.edu/2008/02/488-nm-a-review-of-facs-technology-and-its-application-in-biological-
research/#.V-s5GFt96Un>.
FACS, M. 2016, Magnetic-activated cell sorting vs. FACS. [online] Biology.stackexchange.com, viewed 22 September 2016,
<http://biology.stackexchange.com/questions/31482/magnetic-activated-cell-sorting-vs-facs>.
Kühl, A. A., Pawlowski, N. N., Grollich, K., Blessenohl, M., Westermann, J., Zeitz, M., Loddenkemper, C. & Hoffmann, J. C. 2009, ‘Human
peripheral γδ T cells possess regulatory potential’, Immunology, 128(4), 580–588. http://doi.org/10.1111/j.1365-2567.2009.03162.x](https://image.slidesharecdn.com/applicationoffluorescenceactivated-cellsortingfacs-160929155435/85/Application-of-Fluorescence-Activated-Cell-Sorting-FACS-in-separation-of-different-populations-of-cells-from-a-mixed-community-12-320.jpg)
![ Lauren, N. 2007, Fluorescence-Activated Cell Sorting in Microfluidic Devices. 1st ed. [ebook] Boulder: University of Colorado, p.13, viewed 24
September 2016, <http://www.colorado.edu/physics/Web/reu/Projects/Projects%202007/Lauren%20Nicolaisen.pdf>.
Lawrence, J. 2016, Scientists Discover Biomarkers for Diagnosing Idiopathic Pulmonary Fibrosis, online, Naturalsciencenews.com, viewed 28
September 2016, <http://naturalsciencenews.com/2016/09/02/scientists-discover-biomarkers-for-diagnosing-idiopathic-pulmonary-fibrosis/>.
Mattanovich, D. & Borth, N. 2006, ‘Applications of cell sorting in biotechnology’, Microbial Cell Factories, 5, 12. http://doi.org/10.1186/1475-
2859-5-12
Robertson, S. 2010, Fluorescence-Activated Cell Sorting, viewed 21 September 2016, < http://www.news-medical.net/life-
sciences/Fluorescence-Activated-Cell-Sorting.aspx>.
Roest, K. 2007, ‘Microbial community analysis in sludge of anaerobic wastewater treatment systems’, PhD thesis, Wageningen University,
Netherlands.
References](https://image.slidesharecdn.com/applicationoffluorescenceactivated-cellsortingfacs-160929155435/85/Application-of-Fluorescence-Activated-Cell-Sorting-FACS-in-separation-of-different-populations-of-cells-from-a-mixed-community-13-320.jpg)
Application of Fluorescence Activated-Cell Sorting (FACS) in separation of different populations of cells from a mixed community
- 1. Application of Fluorescence Activated-Cell Sorting (FACS) -Separation of different populations of cells from a mixed community Presented by : Goh Mei Ying (0317999) Lim Tze Shien (0323020) Muhammad Uzair (0321618) Nur Nabihah Mohamat (0318664) Ting Sing Hong (0317799)
- 2. Introduction Fluorescence Activated-Cell Sorting Enables a mixture of different cells to be sorted one by one into one or more containers. Cells are sorted according to their specific light scattering and fluorescent characteristics (Robertson 2010). (Blogs.sun.ac.za n.d.) The ways to display the FACS data collected by computer: (Robertson 2010)
- 3. (Bio.davidson.edu 2001) Cells (by tagging to an antibody linked to fluorescent dye) are passed into the middle of a fast flowing liquid stream Vibration is applied to break the stream up into droplets Laser light excites the dye which emits a colour of light The colored light is detected by a photomultiplier tube or a light detector An electrical charging ring is positioned An electrical charge is applied to the stream and the newly formed drop will form with charge Charged drop will be then deflected left or right by charged electrodes and into waiting sample tube (Robertson 2010)
- 4. Discussion Application used to separate different populations of cells from a mixed community where it allows physical enrichment or isolation even of yet uncultured organisms that can be used for subsequent molecular genetic studies and cultivation (Roest 2007). Park et al. (cited in Roest 2007) used this technique for analysis of activated- sludge and Yellowstone Lake hydrothermal vent samples and he detected various unknown bacterial species which were not detectable in the previous original sample due to low relative abundance and this limitation could be overcome by the application of FACS.
- 5. Discussion Application Separation & isolation of γδ T cells from a population of lymphocytes. Why study γδ T cells ? Deficiency of γδ T cells aggravates colitis in animal models. Suggesting that they possesses regulatory properties in secretion of cytokines. γδ T cells specific subset of T cells, play a prominent role in recognizing lipid antigens, may be triggered by alarm signals, such as heat shock proteins. Cell sample are stained with fluorescent antibody which binds specifically to surface markers on γδ T cells. (Kühl et al. 2009) (Dartmouth Undergraduate Journal of Science 2008)
- 6. Discussion Application Screening for intracellular and secreted proteins Cells with high secretion rate can be sorted and subcloned. Plasmids are isolated and retransformed into the host strain. (Mattanovich & Borth 2006)
- 7. Idiopathic Pulmonary Fibrosis (IPF) Diagnostics Idiopathic – any disease/condition that arise spontaneously with an unknown cause. Pulmonary Fibrosis – scar tissue forms in lungs. Lung tissue become stiff and scarred. S & S – dry cough & trouble breathing.
- 8. Idiopathic Pulmonary Fibrosis (IPF) Diagnostics Myeloid-Derived Suppressor Cells (MDSC) Suppress immune system Tissue remodeling angiogenesis aggressive cancer (Bronte and Gabrilovich 2016) Fluorescence Activated Cell Sorting (FACS) helps to determine quantity of MDSC in blood samples. Higher number of MDSC found in IPF patients. MDSC as biomarkers. (Lawrence 2016)
- 9. Sorter is mainly for the use on a large population of cells causing the recovery rate is fairly slow (Lauren 2007). There is a tradeoff between the sorting speed, purity rate and recovery state (Lauren 2007). Currently, traditional FACS machine is very large and space consuming but it is the ideal size for its function (Lauren 2007). In modern cancer and immunology research, it is considered expensive due to the inevitable process to recover all the cells from the sorter with maximum possible purity rate (FACS 2016). FACS is limited to its specific purpose and prolonged process, more than 6 hours compared to magnetic cell sorting type (eg. FACS only enable one cell to pass through the laser focus at a time) (Catherine, Brian T and Timothy C 2010). Requires debulking process first (Catherine, Brian T and Timothy C 2010). Limited number of fluorophore-conjugated antibody reagent for clinical processing (Catherine, Brian T and Timothy C 2010). Direct and indirect method for Immunofluorescence are correlated to each other to give a better results (Abcam.com 2016). Challenging to detect low abundance proteins even with indirect methods. (eg. Biotinylated antibodies offer an extra layer for increased signal amplification.) (Abcam.com 2016). Main Challenges & Limitation
- 10. Table 1: Comparison of cell isolation approaches by surface antigen-based Figure 1:Traditional cell sorting machine (Lauren 2007).
- 11. Conclusion Function of FACS To sort cells according to their specific light scattering and fluorescent characteristics. Applications Separation of different populations of cells from a mixed community Separation & isolation of γδ T cells from a population of lymphocytes Screening of intracellular and secreted protein Current development Determination of quantity of MDSC in blood samples of IPF patients. Challenges & limitation Slow recovery rate Space consuming Expensive; Long processing hours Requires debulking process Limited number of fluorophore-conjugated antibody reagent Challenging to detect low abundance proteins
- 12. References Abcam.com 2016, Direct vs indirect immunofluorescence | Abcam., online, viewed 24 September 2016, <http://www.abcam.com/secondary- antibodies/direct-vs-indirect-immunofluorescence>. Bio.davidson.edu 2001, FACS Methodology, viewed 21 September 2016, <http://www.bio.davidson.edu/courses/genomics/method/facs.html>. Blogs.sun.ac.za n.d., BD FACS Calibur », viewed 21 September 2016, <http://blogs.sun.ac.za/fmc/bd-facs-calibur/>. Bronte, V. and Gabrilovich, D. 2016, Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards. Nature Communications, 7, p.12150. Catherine, M., Brian T, F. and Timothy C, F. 2010, Fluorescence-Activated Cell Sorting for CGMP Processing of Therapeutic Cells. 1st ed. [ebook] Sparks: BD biosciences, p.7, viewed 23 September 2016, <https://www.researchgate.net/profile/Timothy_Fong2/publication/228470167_FluorescenceActivated_Cell_Sorting_for_CGMP_Processing_of _Therapeutic_Cells/links/55f6ef6e08ae07629dbb159e.pdf>. Dartmouth Undergraduate Journal of Science 2008, 488 nm: A Review of FACS Technology and its Application in Biological Research, online, viewed 23 September 2016, < http://dujs.dartmouth.edu/2008/02/488-nm-a-review-of-facs-technology-and-its-application-in-biological- research/#.V-s5GFt96Un>. FACS, M. 2016, Magnetic-activated cell sorting vs. FACS. [online] Biology.stackexchange.com, viewed 22 September 2016, <http://biology.stackexchange.com/questions/31482/magnetic-activated-cell-sorting-vs-facs>. Kühl, A. A., Pawlowski, N. N., Grollich, K., Blessenohl, M., Westermann, J., Zeitz, M., Loddenkemper, C. & Hoffmann, J. C. 2009, ‘Human peripheral γδ T cells possess regulatory potential’, Immunology, 128(4), 580–588. http://doi.org/10.1111/j.1365-2567.2009.03162.x
- 13. Lauren, N. 2007, Fluorescence-Activated Cell Sorting in Microfluidic Devices. 1st ed. [ebook] Boulder: University of Colorado, p.13, viewed 24 September 2016, <http://www.colorado.edu/physics/Web/reu/Projects/Projects%202007/Lauren%20Nicolaisen.pdf>. Lawrence, J. 2016, Scientists Discover Biomarkers for Diagnosing Idiopathic Pulmonary Fibrosis, online, Naturalsciencenews.com, viewed 28 September 2016, <http://naturalsciencenews.com/2016/09/02/scientists-discover-biomarkers-for-diagnosing-idiopathic-pulmonary-fibrosis/>. Mattanovich, D. & Borth, N. 2006, ‘Applications of cell sorting in biotechnology’, Microbial Cell Factories, 5, 12. http://doi.org/10.1186/1475- 2859-5-12 Robertson, S. 2010, Fluorescence-Activated Cell Sorting, viewed 21 September 2016, < http://www.news-medical.net/life- sciences/Fluorescence-Activated-Cell-Sorting.aspx>. Roest, K. 2007, ‘Microbial community analysis in sludge of anaerobic wastewater treatment systems’, PhD thesis, Wageningen University, Netherlands. References
- 14. ~ Thank you ~
- 15. ~ Q & A ~
Editor's Notes
- #3: Fluorescence-Activated Cell Sorting is a flow cytometer that enables a mixture of different cells to be sorted one by one into one or more containers according to their specific light scattering and fluorescent characteristics. Here are the ways to display the data. First method is best to display the data if no cells are labeled both colors. For the second methods, we cant to count for each spot, just can see the relative density. Top right: no cells labelled both colors, bottom left: cells that unlabelled.
- #4: Cells(by tagging to an antibody linked to fluorescent dye) are passed into the middle of a fast flowing liquid stream to ensure that they are well separated. Vibration is applied to break the stream up into droplets in such a way one cell is presented per drop. Laser light excites the dye which emits a color of light The colored light is detected by a photomultiplier tube or a light detector An electrical charging ring is positioned An electrical charge is applied to the stream and the newly formed drop will form with charge Charged drop will be then deflected left or right by charged electrodes and into waiting sample tube
- #6: Deficiency in γδ T cells aggravates colitis in animal models suggesting that γδ T cells have regulatory properties. Therefore, proliferation, suppression and cytokine secretion of human γδ T cells were determined in vitro.
- #7: When sorting is used for the selection of over-producing cells, there are three main objectives: to reduce the work load necessary to find over-producers, (2) to reduce the time required and (3) to find the cells with the highest possible production rates, ideally combined with other advantageous properties Immunofluorescence based screening methods for intracellular and secreted proteins. A: Screening for secreted proteins, developed for mammalian cells. A product specific catching antibody is immobilized on the cell surface, which binds an amount of product proportional to the secretion rate. After staining with a product specific antibody and a secondary antibody, cells with high secretion rate can be sorted and subcloned. B: Screening system for intracellular, plasmid encoded proteins, developed for E. coli. Cells are fixed with ethanol and stained with a product specific antibody and a secondary antibody After sorting, plasmids are isolated and retransformed into the host strain.