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)

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)

(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)

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.

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)

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)

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.

Idiopathic Pulmonary Fibrosis (IPF) Diagnostics

Myeloid-Derived Suppressor Cells (MDSC) Suppress immune systemTissue 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)

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

Table 1: Comparison of cell isolation approaches by surface antigen-basedFigure 1:Traditional cell sorting machine (Lauren 2007).

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

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

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

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