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FACS and MACS with their applications in biological research.

Flow cytometry (FACS) and magnetic activated cell sorting (MACS) are techniques used to analyze and separate cells based on their physical and chemical characteristics. FACS uses lasers to detect cell properties and sort cells into containers one by one, while MACS uses magnetic microbeads attached to cells to separate them in high gradient magnetic fields. These techniques have various applications in research including identifying stem cells, characterizing cancer cells, studying cell cycles, and isolating cell populations for further analysis.

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Application of FACS and MACS in biological research Deepak Agarwal FBT-02-06 Major Advisor Dr. Aparna Chaudhari
Introduction  Flow cytometry (FCM) is a technique used to rapidly detect and count microscopic particles such as cells, suspended in a stream of fluid.  It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second.  FCM is used in the diagnosis of health disorders, and has many applications in both research and clinical practice.  It is used To Count T cell and B cell  An impedance-based flow cytometry device was patented in 1953; the first fluorescence-based flow cytometry device was developed in the late 1960’s  Protein expression  Protein localization  To differenciate cancerous cells  To analysis of cell cycle, DNA content etc….
Fluorescence-activated cell sorting  Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry.  It provides a method for sorting a heterogeneous mixture of cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell.  It provides fast, objective and quantitative recording of signals from individual cells as well as physical separation of cells of particular interest.  The first cell sorter was invented in 1965 by Mack Fulwyler (1965)  Technique expanded by Len Herzenberg – coin term “FACS”  Invention in late 1960s by Bonner, Sweet, Hulett, Herzenberg  The commercial machines introduction- Becton Dickinson Immunocytometry Systems in the early 1970s  It can separate one fluorescent cell from a population of 1000 unlabelled cells.
Principle  One or more beams of light (usually laser light) is directed onto a hydrodynamically- focused stream of fluid.  A number of detectors are placed at the intersection of the stream with the light beam, to detect scattered light (forward scatter or FSC, in line with the light beam, and side scatter or SSC, perpendicular to it) and one or more fluorescent detectors.  Each suspended particle –from 0.2 to 150 micrometers- passing through the beam scatters the light, and fluorescent chemicals found in the particle or attached to it may be excited, emitting light at a longer wavelength than the light source.  This combination of scattered and fluorescent light is recorded by the detectors and, by analyzing changes in brightness at each detector, information about the physical and chemical structure of each individual particle is obtained.  FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (shape of the nucleus, the amount and type of cytoplasmatic granules or the membrane roughness).  Modern flow cytometers are able to analyze several thousand particles every second, in "real time", and can actively isolate particles having specified properties.
INSTRUMENTATION
Fluidics – direct liquid stream containing particles through focused laser beam Vibrating nozzle- it breaks the cell suspension into fine droplets. Laser – light source to focus light Collection optics and filters – detect light signals coming from particles Electronics/computer – convert light signals to voltage and digital output
 The fluidic system often uses air pressure regulation for stable operation and consists of at least one sheath line and a sample line feeding the flow cell.  As the sample enters the flow cell chamber, the outer, faster flowing sheath fluid hydrodynamically focuses this fluid into a narrow core region within the jet and presents a single file of particles to excitation sources.  This geometry provides increased positioning accuracy at the laser interrogation point for consistent excitation irradiance and greatly reduced particle blockage of the flow cell Fluidics • The fluidic system is used to transport particles from a random three-dimensional sample suspension to an orderly stream of particles.
Optics  Optics are central to flow cytometry for the illumination of stained and unstained particles and for the detection of scatter and fluorescent light signals.  Commonly used are lamps (mercury, xenon); high-power water-cooled lasers (argon, krypton, dye laser); low-power air-cooled lasers (argon (488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet) resulting in light signals
Light Collection Laser beam Side scatter - 90⁰ Forward scatter
Optical Filters  Once the fluorescence light from a cell has been captured by the collection optics, the spectral component of interest for each stain must be separated spatially for detection.  This separation of wavelengths is achieved using dichroic (45 degree) and emission (normal incidence) filters.  Longpass filters permit longer wavelength transmission, while shortpass filters allow shorter wavelength transmission.  Bandpass filters only allow a selected wavelength band of interest to be transmitted while blocking unwanted wavelengths.
Detectors  Silicon photodiodes and photomultiplier tubes (PMTs). Electronics  As a particle of interest passes through the focus, fluoresces and is detected by a photodetector, an electrical pulse is generated and presented to the signal processing electronics.  An amplification system - linear or logarithmic  A computer for analysis of the signals.
Process of operation  The cell suspension containing the cells labeled with fluorescent dye is directed into a thin stream so that all cells pass in a single file.  The stream emerges from nozzle vibrating at a some 40000 cps.  It breaks the stream into 40000 droplets per sec.  Laser beam is directed at the stream just before it breaks up into droplets.  As each labeled cell passes through the beam, its resulting fluorescence is detected by a photocell.  If cell is fluorescent then it given a charge +ve or –ve.  The droplets retain this charge as they pass between a pair of charged metal plates.  Positively charged cells are attracted by a negatively charged plate vice versa.  Uncharged droplets doesn’t deviate and it pass straight into the third container and discarded later.
 Cell scatters light in all directions when passes through the laser.  Two types of scatters : Forward Scatter Side Scatter
• Low angle scattering (upto 20o from beam of axis). • Detector converts intensity of light into voltage. • Blocking bar is placed in front of detector. • In absence of cell light falls on blocking bar so no voltage. • When cell passes through beam scattered light falls on detector and voltage is measured. FORWARD SCATTER ABSENCE OF CELL IN BEAM PRESENCE OF CELL IN BEAM
CELL SIZE AND VOLTAGE INTENSITY
Electronics convert optic signals to voltage pulse
SIDE SCATTER • Scatter at larger angle. (~90o from beam axis). • Because of granularity and complexity inside the cells. • Focused to the side scatter detection system and detected by separate detectors. • Detection is same as forward scatter detectors.
FLUORESCENT DETECTION • Fluorochrome molecules have Excitation (Accept light at given wavelength) and Emission (Re-emit light at higher wavelength) processes. 41 3 1. Light is absorbed by fluorochrome and electrons become excited. 2. Excited electrons migrate from Resting (Ground) state to Excited state. 3. Within 10 nanoseconds it releases some of absorbed energy as heat and fall lower to more stable level. 4. Electron steadily moves back to Ground state by releasing energy as Fluoroscence.STOKES SHIFT 2
FLUORESCENT DETECTION • Absorbed Energy (Eexcitation) > Released Energy (Eemission) • Stokes Shift = Eexcitation - Eemission. • Value of Stokes Shift determines quality of fluorochrome. • Higher the value of Stokes Shift easier the detection. • Fluorescent Probes directly target the interested cells so readily chosen for flowcytometric analysis. • More parameters can be detected one at a time if more fluorochromes are used. • Generally Tandem Dyes are used for detection. •E.g. Alexa Fluor 488, Phycoerythrin, APC Cy 7.
FLUORESCENT DETECTION FLUORESCENT LABELLED Ab CELL • Fluorophores are attached to the cell surface or inside the cell. • Fluorescent signals are emitted when cell passes through laser light (635 nm, 488 nm). • Signals are collected by various mirrors and filters and detected by Photodiodes or PMTs.
Quantifying FACS Data FACS data collected by the computer can be displayed in two different ways
Here we see a different way to display the same data. • The X-axis plots the intensity of green fluorescence while the Y-axis plots the intensity of red fluorescence. • The individual black dots represent individual cells and we are not supposed to count the dots but just look at the relative density of dots in each quadrant. Cont.….
Magnetic Activated Cells Sorting(MACS)  A flexible, fast and simple magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers.  Cells stained sequentially with biotinylated antibodies, fluorochrome-conjugated avidin, and superparamagnetic biotinylated-microparticles (about 100 nm diameter) are separated on high gradient magnetic (HGM) columns.  More than 109 cells can be processed in about 15 min.  The simultaneous tagging of cells with fluorochromes and very small, invisible magnetic beads makes this system an ideal complement to flow cytometry.  Light scatter and fluorescent parameters of the cells are not changed by the bound particles.  Magnetically separated cells can be analyzed by fluorescence microscopy or flow cytometry or sorted by fluorescence- activated cell sorting without further treatment.  Magnetic tagging and separation does not affect cell viability and proliferation.
Cont.…  Bound large particles have disadvantages.  Magnetic microparticles (diameterparticl(e ( 0.5 pm) can have a variety of superior characteristics compared to larger particles.  Their major disadvantage is the small magnetic moment, resulting in long separation times in magnetic fields.  To overcome this disadvantage, Molday and Molday have suggested a combination of small superparamagnetic microparticles and high gradient magnetic (HGM) fields with Yields Relatively High Purity Cell Populations.
MACSŽ Technology  3 Components Involved  MACS MicroBeads  MACS Column  MACS Separator  MACS MicroBeads  50 nm(superparamagnetic particles) in size  Biodegradable  Conjugated to monoclonal antibodies  MACS Column  Generates strong magnetic field
Direct Labeling  Advantages  Fast  Yields High Purity Cell Populations  Disadvantages  Binding of antibody to surface marker may activate cell  Some cell populations may require multiple markers to be identified Indirect Labeling • Useful in cases with dim surface marker expression • Allows for amplification of signal • Secondary antibody recognizes • Biotin • FITC • Fc portion of primary antibody • Streptavidin instead of secondary antibody • Recognizes biotin on primary antibody Bead Monoclonal Ab Labeling Of Cells With Beads
MACS separation unit
MiniMACS Separator Magnet MS Column
Magnetic Separation High Gradient Magnetic Field Trapped Labeled Cells Cells Of Interest
Positive selection Negative selection,cell Depletion  Positive selection: desired cell population is magnetically labeled and isolated as the retained cell fraction.  Negative selection: Depletion of undesired cells. Non-target cells are magnetically labeled and depleted from the cell mixture. The flow through contains desired cell fraction.  Useful In Following Cases  Removal of Unwanted Cells  No specific antibody is available for target cell  Binding of antibody to target cell results in activations.
Automated MACS separation
Dynal versus MACS beads
Comparison FACS versus MACS
Applications of FACS and MACS Utility in assisted reproduction Isolation Of CD4+ Cells Characterization of multidrug resistance (MDR) cells Multiplex protein assay cytometric bead array Cell Cycle Analysis Analysis of Intracellular Ion Concentration Differentiating cancerous cells Chromosome analysis and sorting Measuring cell membrane permeability Stem cell identification
Magnetic activated cell sorting (MACS): Utility in assisted reproduction  Assisted reproductive techniques (ART) have now been extensively incorporated in the management of infertile couples.  Conventional semen analysis .  Sperm apoptosis has been heavily linked to failures in reproductive techniques. One of the earliest changes shown by apoptotic spermatozoa is externalization of phosphatidyl serine.  Magnetic activated cell sorting (MACS) is a novel sperm preparation technique that separates apoptotic and non-apoptotic spermatozoa based on the expression of phosphatidylserine.  By separating the apoptotic spermatozoa it has also improved the success rates of assisted reproduction techniques.
How does it work???  MACS technology uses annexin V-conjugated superparamagnetic microbeads (50 nm) to separate nonapoptotic spermatozoa from those with deteriorated plasma membranes and externalization of PS.  Depending on Ca2+, PS has a high affinity for annexin V, which is 35-36 kDa phospholipid binding protein.  Annexin-V does not have the ability to pass the intact sperm membrane, so the annexin-V binding to spermatozoa characterizes disturbed integrity of the sperm membrane.  Thus annexin enables the identification of cells with altered membrane integrity Based on annexin binding and subsequent magnetic separation 2 fractions are obtained.  annexin-negative(unlabeled-intact membrane; non-apoptotic) and annexin positive (labeled- altered membrane; apoptotic).
Isolation Of CD4+ Cells Use CD4(L3T4) Microbeads.
Charactrization of multidrug resistance (MDR) cells • The multidrug resistance (MDR) phenotype is associated with the overexpression of members of the ATP-binding cassette family of proteins. These MDR transporters are expressed at the plasma membrane. • The multidrug resistance protein 1 (MRP1), P-glycoprotein, and the breast cancer resistance protein are each present in a subcellular compartments and perinuclear region. • Fluorescence-activated cell sorting detection of the three most studied transporters: P-glycoprotein, multidrug resistance-associated proteins, and breast cancer resistance protein.
Cont.…  Detection of P-glycoprotein using the antibody MRK16  Detection of MDR1 Expression using antibody CD243
Multiplex protein assay cytometric bead array  A multiplex assay is a type of assay that simultaneously measures multiple analytes (dozens or more) in a single run/cycle of the assay.  Multiplex assays are widely used in functional genomics experiments that endeavor to detect or to assay the state of all biomolecules of a given class (e.g., mRNAs, proteins) within a biological sample.  Multiplexing offers several distinct advantages over single-plex assays
Cytometric Bead Array (CBA) assays  In bead-based assays, immunodetection occurs not on the flat surface of a membrane or microtiter plate but on micron-sized spheres.  Each bead contains a unique blend of fluorophores that acts as a signature and is associated with a single analyte—bead identifier 1 corresponds to IL-2, identifier 2 to IL-4 and so on.  Multiplexing is accomplished by combining different bead sets (with associated capture antibodies) into one master mix and incubating that mix with each sample in a microtiter plate.  Several multiplexed bead systems are available. Cytometric Bead Array (CBA) assays, which can be multiplexed to 30 analytes.
Cell Cycle Analysis  One of the most common uses of FACS is to analyze the cell cycle of mammalian cells.  FACS can measure the deoxyribonucleic acid (DNA) content of individual cells at a rate of several thousand cells per second and thus conveniently reveals the distribution of cells through the cell cycle.  G2/M-phase cells have twice the amount of DNA as G1/G0-phase cells, and S- phase cells contain varying amounts of DNA between that found in G1 and G2 cells.  Furthermore, each subpopulation can be quantified
Analysis of Intracellular Ion Concentration  An alteration in intracellular calcium concentration is one of the most common second messenger responses now known in mammalian cells.  The vast majority of changes in intracellular calcium is extremely rapid and occurs within a nanosecond timescale.  The recent development of a number of new fluorescent probes makes it possible to measure the concentrations of various intracellular free ions in single living cells.  Among these ions are calcium, magnesium, sodium, potassium, and hydrogen (pH).  Using the dyes Indo-1 AM, Fluo-3, and Fura Red AM to measure intracellular calcium concentration.
Differentiating cancerous cells  Some of the determinants of tumor response seem to be expressed at the cellular level in terms of degree of tumor cell differentiation.  Quantitative cytology in the form of flow cytometry has greatly advanced the objective elucidation of tumor cell heterogeneity by using probes that discriminate tumor and normal cells and assess differentiative as well as proliferative tumor cell properties.  Among phenotypic tumor cell markers, surface membrane antigens have been extensively studied in lymphoid and myeloid neoplasms by the use of hybridoma-generated monoclonal antibodies, which have recently also found in vitro and in vivo therapeutic application.
Cont.….  FACS is used for the identification and effective isolation of CSCs .  Similar to hematopoietic stem cells or stem cells from other tissues, measurements of specific cluster of differentiation (CD) surface markers and stem cell-specific metabolic activities have been used for the characterization of CSCs.  The list of CD markers found to be associated with CSCs is extensive and includes many markers used for the identification of nonmalignant hematopoietic or mesenchymal stem cells and other special markers like the B-cell marker CD20 for melanomas .
Chromosome analysis and sorting  Conventional karyotyping involves the cytogenetic analysis of a metaphase spread.  FACS from monodispersed suspension of chromosomes.  A good chromosome preparation is derived from a semi-confluent cell culture which is an exponential phase of growth.  DNA dyes - chromomycin A3 which stains CG-rich regions of DNA and Hoechst 33258 which stains AT-rich regions.  The chromosomes then separate according to their DNA content (size) and base pair composition.
Measuring cell membrane permeability  A reliable and rapid test to detect cytotoxic chemicals which affect cell membranes.  Fluorescein diacetate freely penetrates intact cells.  On the other hand, ethidium bromide is known to be excluded from the intact cell.  The combination of both fluorochromes results in counter-staining: intact cells fluoresce green (cytoplasm) and membrane-damaged cells fluoresce red (nucleus and RNA).
Cont.…. Human ovarian carcinoma cell line labelled with FDA and PI(propidium iodide). The viable cells are fluorescein +ve, PI -ve and the dead cell fluorescein -ve, PI +ve. Green: live cells. Red: dead cells. Some of the blue cells are probably apoptotic.
Some other Applications:  Counting T cell and B cell  Protein expression and localization.  Transgenic products in vivo (particularly the green fluorescent protein (GFP)  Immunophenotyping by related fluorescent cell surface antigens (Cluster of differentiation (CD) markers), intracellular antigens (various cytokines, secondary mediators, etc.), nuclear antigens.  Enzymatic activity, pH, apoptosis.  Measurement of DNA degradation, mitochondrial membrane potential, permeability changes.
References
 For Analysis of Apoptosis by Flow Cytometry." Manual of Clinical Laboratory Immunology, 5th edn. Ed. Noel R. Rose, Everly Conway deMacario, James D. Folds, H. Clifford Lane, Robert M. Nakamura. Washington, D.C.: American Society for Microbiology, 1997. 334-343.  Blue, Julia; French, T.W. "Hematology Atlas." 3 March 1997. http://drylab1.vet.cornell.edu/clinpathmodules/heme1/lymph.htm (06 October 1997).  Janeway, Charles A.; Travers, Paul. Immunobiology, 3rd Edn. New York: Current Biology Limited/Garland Publishing, Inc. 1997.  Kantor, Aaron B.; Merrill, Cynthia E.; Hillson, Jan L. "Construction of cDNA from Single Unstimulated Mouse B Lymphocytes: Method and Application to the Study of Expressed Antibody Repertoires in FACS- Sorted Murine B Cell Subsets." Immunochemistry and Molecular Immunology, vol. 1, 5th edn. Ed. Leonore A. Herzenberg, Donald M. Weir, Leonard A. Herzenberg, and Caroline Blackwell. Oxford: Blackwell Scientific Publications, 1996. 13.1-13.6.  Kidd, Pamela G.; Nicholson, Janet K.A. "Immunophenotyping by Flow Cytometry." Manual of Clinical Laboratory Immunology, 5th edn. Ed. Noel R. Rose, Everly Conway de Macario, James D. Folds, H. Clifford Lane, Robert M. Nakamura. Washington, D.C.: American Society for Microbiology, 1997. 229- 244.  Lefrancois, Leo; Olson, Sarah; Pizzo, Gene. "Flow Cytometry: How Cytometry Works." n.d.. http://www9.uchu.edu/grad/immuno/pizzo/how.htm (06 October 1997).  Stadler, Beda M. "Flow Cytometry Laboratory." 27 December 1996. http://ubeclu.unibe.ch/insel/cytindex.htm (08 October 1997).  "What Is Flow Cytometry?" 07 October 1997. http://www4.umdnj.edu/cytoweb/intro.htm (07 October 1997).
Cont.…  Gelman R, Wilkening C. Analysis of quality assessment studies using CD45 for gating lymphocytes for CD3+4+%. Cytometry 2000;42:1-4.  Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines for CD34+ cell determinations by flow cytometry. J Haematotherapy 1996;5:213-26.  Mandy FF, Bergeron M, Minkus T. Evolution of leukocyte immunophenotyping as influenced by the HIV/AIDS pandemic: a short history of the development of gating strategies for CD4+ T cell enumeration. Cytometry 1997;30:157-65.  Robinson G, Morgan L, Evans M, McDermott S, Pereira S, Wansbrough-Jones M et al. Effect of the type of haematology analyser on CD4 count. Lancet 1992;340(ii):485.  Barnett D, Granger V, Whitby L, Storie I, Reilly JT. Absolute CD4+ T lymphocyte and CD34+ stem cell counts by single platform flow cytometry: the way forward. British Journal Haematology 1999;106:1059-62.  Bergeron M, Lustyik G, Ding T, Nicholson J, Janossy G, Shapiro H et al. When do non-volumetric flow cytometers become volumetric? Time can tell how absolute your instrument is about absolute counts. Cytometry 2003;52B:37-9.  Brando B, Barnett D, Janossy G et al. Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. Cytometry 2000;42:327-46.  Mandy FF, Nicholson JKA, McDougal JS. Guidelines for performing single platform absolute CD4+ T cell determinations with CD45 gating for persons infected with human immunodeficiency virus. MMWR Jan 31 2003;52,RR-2.  Storie I, Sawle A, Goodfellow K, Whitby L, Granger V, Reilly JT et al. Flow rate calibration: a novel approach for performing absolute cell counts. Cytometry 2003;55B:1-7.
Cont.…  Larry A. Sklar and David A. Finney (1982) Analysis of Ligand-Receptor Interactions with the Fluorescence Activated Cell Sorter cytometry Pages 161-165 Vol. 3, No. 3  Anne Yen-Chen Fu(2002) Microfabricated Fluorescence-Activated Cell Sorters (FACS) for Screening Bacterial Cells  Centre for environmental health sciences, university of Montana. Guidelines for FACS  Lauren Nicolaisen, University of Colorado, Boulder. Fluorescence-Activated Cell Sorting in Microfluidic Devices  Leonard A Herzenberg, David Parks, Bita Sahaf, Omar Perez, Mario Roederer and Leonore A. Herzenberg (2002) The History and Future of the Fluorescence Activated Cell Sorter and Flow Cytometry: A View from Stanford Clinical Chemistry 48:10 1819–1827  R. Ian Freshney -Culture of animal cells- A mannual of basic technique p 244-245  Tortora, Funke and Case: Microbiology – An introduction  http://www.mdanderson.org/education-and-research/resources-for-professionals/scientific-resources/core- facilities-and-services/flow-cytometry-and-cellular-imaging-core- facility/sops/index.html#6.MultidrugResistanceAssays  Altan, N., Chen, Y., Schindler, M., and Simon, S.M. (1998). Defective acidification in human breast tumor cells and implications for chemotherapy. J. Exp. Med. 187, 1583–1598. [PMC free article][PubMed]  Cabrita, M.A., Hobman, T.C., Hogue, D.L., King, K.M., and Cass, C.E. (1999). Mouse transporter protein, a membrane protein that regulates cellular multidrug resistance, is localized to lysosomes. Cancer Res. 59, 4890–4897. [PubMed]
Cont….  Cole, S.P., Chanda, E.R., Dicke, F.P., Gerlach, J.H., and Mirski, S.E. (1991). Non-P-glycoprotein-mediated multidrug resistance in a small cell lung cancer cell line: evidence for decreased susceptibility to drug- induced DNA damage and reduced levels of topoisomerase II. Cancer Res.51, 3345–3352. [PubMed]  Cole, S.P.C., Bhardwaj, G., Gerlach, J.H., Mackie, J.E., Grant, C.E., Almquist, K.C., Stewart, A.J., Kurz, E.U., Duncan, A.M.V., and Deeley, R.G. (1992). Overexpression of a transporter gene in a multidrug- resistant human lung cancer cell line. Science 258, 1650–1654. [PubMed]  Cole, S.P.C., Sparks, K.E., Fraser, K., Loe, D.W., Grant, C.E., Wilson, G.M., and Deeley, R.G. (1994). Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells. Cancer Res. 54, 5902–5910. [PubMed]  Doyle, L.A., Yang, W., Abruzzo, L.V., Krogmann, T., Gao, Y., Rishi, A.K., and Ross, D.D. (1998). A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc. Natl. Acad. Sci. USA 95, 15665– 15670. [PMC free article] [PubMed]  Farkas, D.L., Wei, M., Febbroriello, P., Carson, J.H., and Loew, L.M. (1989). Simultaneous imaging of cell and mitochondrial membrane potentials. Biophys. J. 56, 1053–1069. [PMC free article][PubMed  Horan PK, Wheeless LL. Quantitative Single Cell Analysis and Sorting. Science. 1977;198:149–157.[PubMed  Earley MC, Vogt RF, Shapiro HM, Mandy FF, Kellar KL, Bellisario R, Pass KA, Marti GE, Stewart CC, Hannon WH. Report from a Workshop on Multianalyte Microsphere Arrays. Cytometry. 2002;50:239–242. [PubMed]  Pang S, Smith J, Onley D, Reeve J, Walker M, Foy C. A Comparability Study of the Emerging Protein Array Platforms with Established ELISA Procedures. J Immunol Methods. 2005;302:1–12. [PubMed]  http://www.biocompare.com/Editorial-Articles/41806-Multiplexed-Protein-Assays/
Cont….  Baker PF, Hodgkin AL, Ridgway EB. Depolarization and calcium entry in squid gian axons. J Physiol 1971;218:709–755.  Jackson VM, Trout SJ, Brain KL, Cunnane TC. Characterization of action potentialevoke calcium transients in mous postganglionic sympathetic axon bundles J Physiol 2001;15:3–16.  Murphy A, Vines A, McBean GJ. Stimulation of EAAC1 in C6 glioma cells by storeoperate calcium influx. Biochim Biophy Acta 2009;1788:551–558.  Tsutsumi M, Inoue K, Denda S, Ikeyama K, Goto M, Denda M. Mechanical-stimulation  Venere M, Fine HA, Dirks PB, Rich JN. Cancer stem cells in gliomas: Identifying and understanding the apex cell in cancer’s hierarchy. Glia 2011;59:1148–1154.  Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM. Cancer stem cells—Perspectives on current status and future directions: AACRWorkshop on cancer stem cells. Cancer Res 2006;66:9339–9344.  Lobo NA, Shimono Y, Qian D, Clarke MF. The biology of cancer stem cells. Annu Rev Cell Dev Biol 2007;23:675–699.  Molyneux G, Geyer FC, Magnay FA, McCarthy A, Kendrick H, Natrajan R, Mackay A, Grigoriadis A, Tutt A, Ashworth A, et al. BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells. Cell Stem Cell 2010;7:403–417.
Cont…  High Gradient Magnetic Cell Separation With MACS1 Stefan Miltenyi, Werner Muller, Walter Weichel, and Andreas Radbruch Institute for Genetics, University of Cologne, D5000 Cologne, West Germany ,Received April 6, 1989; accepted July 24, 1989.  Antoine JC, Ternynck T, Rodrigot M, Avrameas S: Lymphoid fractionation on magnetic polyacrylamine agarose beads. Immunochemistry 15:443-447, 1978.  Gold ER, Fundenberg HH: Chromium chloride as a coupling reagent for passive hemagglutination reactions. J Immunol 99: 859-863, 1967.  Groman EV, Sullivan JA, Markinac JE: BioMag-a new support for magnetic affinity chromatography. BioTechniques 156-160, 1985.  Kemshead JT, Ugelstad J: Magnetic separation techniques: Their application to medicine. Mol Cell Biochem 67:ll-18, 1985.  Kronick P, Gilpin W: Use of superparamagnetic particles for separation of cells. J Biochem Biophys Methods 12:73-80, 1986.  Loski CL, Ramm LE, Hammer CH, Mayer MM, Shin ML: Cytolysis of nucleated cells by complement cell death displays multihit characteristics. Proc Natl Acad Sci USA 80:3816-3821, 1983.  Magnetic activated cell sorting (MACS): Utility in assisted reproduction* Kartikeya Makker, Ashok Agarwal† & Rakesh K Sharma Center for Reproductive Medicine, Glickman Urological and Kidney Institute and Obsterics and Gynecology and Women’s Health Institute, Cleveland Clinic, Cleveland, Ohio, 44195 USA.
FACS and MACS with their applications in biological research.

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In this document
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Introduction of FACS and MACS as flow cytometry techniques for biological research.

Overview of FACS methodology and principles, allowing cell sorting and detailed analysis.

Detailed components of flow cytometry equipment including fluidics, optics, and electronics.

Scattering light detection principles, including forward and side scatter in flow cytometry.

Methods of displaying FACS data visually for analysis of cellular properties.

Introduction to MACS, its components, and benefits for cell separation in research. Detailed components of MACS technology and its comparison to FACS for diverse applications.Applications of FACS and MACS including cell isolation and characterization in various fields.

FACS applications in analyzing cell cycles, intracellular ions, cancer cells, and other functions.

Citations and references used throughout the presentation, showcasing foundational research.

FACS and MACS with their applications in biological research.

  • 1.
    Application of FACSand MACS in biological research Deepak Agarwal FBT-02-06 Major Advisor Dr. Aparna Chaudhari
  • 2.
    Introduction  Flow cytometry(FCM) is a technique used to rapidly detect and count microscopic particles such as cells, suspended in a stream of fluid.  It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second.  FCM is used in the diagnosis of health disorders, and has many applications in both research and clinical practice.  It is used To Count T cell and B cell  An impedance-based flow cytometry device was patented in 1953; the first fluorescence-based flow cytometry device was developed in the late 1960’s  Protein expression  Protein localization  To differenciate cancerous cells  To analysis of cell cycle, DNA content etc….
  • 3.
    Fluorescence-activated cell sorting Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry.  It provides a method for sorting a heterogeneous mixture of cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell.  It provides fast, objective and quantitative recording of signals from individual cells as well as physical separation of cells of particular interest.  The first cell sorter was invented in 1965 by Mack Fulwyler (1965)  Technique expanded by Len Herzenberg – coin term “FACS”  Invention in late 1960s by Bonner, Sweet, Hulett, Herzenberg  The commercial machines introduction- Becton Dickinson Immunocytometry Systems in the early 1970s  It can separate one fluorescent cell from a population of 1000 unlabelled cells.
  • 4.
    Principle  One ormore beams of light (usually laser light) is directed onto a hydrodynamically- focused stream of fluid.  A number of detectors are placed at the intersection of the stream with the light beam, to detect scattered light (forward scatter or FSC, in line with the light beam, and side scatter or SSC, perpendicular to it) and one or more fluorescent detectors.  Each suspended particle –from 0.2 to 150 micrometers- passing through the beam scatters the light, and fluorescent chemicals found in the particle or attached to it may be excited, emitting light at a longer wavelength than the light source.  This combination of scattered and fluorescent light is recorded by the detectors and, by analyzing changes in brightness at each detector, information about the physical and chemical structure of each individual particle is obtained.  FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (shape of the nucleus, the amount and type of cytoplasmatic granules or the membrane roughness).  Modern flow cytometers are able to analyze several thousand particles every second, in "real time", and can actively isolate particles having specified properties.
  • 5.
  • 6.
    Fluidics – directliquid stream containing particles through focused laser beam Vibrating nozzle- it breaks the cell suspension into fine droplets. Laser – light source to focus light Collection optics and filters – detect light signals coming from particles Electronics/computer – convert light signals to voltage and digital output
  • 7.
     The fluidicsystem often uses air pressure regulation for stable operation and consists of at least one sheath line and a sample line feeding the flow cell.  As the sample enters the flow cell chamber, the outer, faster flowing sheath fluid hydrodynamically focuses this fluid into a narrow core region within the jet and presents a single file of particles to excitation sources.  This geometry provides increased positioning accuracy at the laser interrogation point for consistent excitation irradiance and greatly reduced particle blockage of the flow cell Fluidics • The fluidic system is used to transport particles from a random three-dimensional sample suspension to an orderly stream of particles.
  • 8.
    Optics  Optics arecentral to flow cytometry for the illumination of stained and unstained particles and for the detection of scatter and fluorescent light signals.  Commonly used are lamps (mercury, xenon); high-power water-cooled lasers (argon, krypton, dye laser); low-power air-cooled lasers (argon (488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet) resulting in light signals
  • 9.
    Light Collection Laser beamSide scatter - 90⁰ Forward scatter
  • 10.
    Optical Filters  Oncethe fluorescence light from a cell has been captured by the collection optics, the spectral component of interest for each stain must be separated spatially for detection.  This separation of wavelengths is achieved using dichroic (45 degree) and emission (normal incidence) filters.  Longpass filters permit longer wavelength transmission, while shortpass filters allow shorter wavelength transmission.  Bandpass filters only allow a selected wavelength band of interest to be transmitted while blocking unwanted wavelengths.
  • 11.
    Detectors  Silicon photodiodesand photomultiplier tubes (PMTs). Electronics  As a particle of interest passes through the focus, fluoresces and is detected by a photodetector, an electrical pulse is generated and presented to the signal processing electronics.  An amplification system - linear or logarithmic  A computer for analysis of the signals.
  • 12.
    Process of operation The cell suspension containing the cells labeled with fluorescent dye is directed into a thin stream so that all cells pass in a single file.  The stream emerges from nozzle vibrating at a some 40000 cps.  It breaks the stream into 40000 droplets per sec.  Laser beam is directed at the stream just before it breaks up into droplets.  As each labeled cell passes through the beam, its resulting fluorescence is detected by a photocell.  If cell is fluorescent then it given a charge +ve or –ve.  The droplets retain this charge as they pass between a pair of charged metal plates.  Positively charged cells are attracted by a negatively charged plate vice versa.  Uncharged droplets doesn’t deviate and it pass straight into the third container and discarded later.
  • 13.
     Cell scatterslight in all directions when passes through the laser.  Two types of scatters : Forward Scatter Side Scatter
  • 14.
    • Low anglescattering (upto 20o from beam of axis). • Detector converts intensity of light into voltage. • Blocking bar is placed in front of detector. • In absence of cell light falls on blocking bar so no voltage. • When cell passes through beam scattered light falls on detector and voltage is measured. FORWARD SCATTER ABSENCE OF CELL IN BEAM PRESENCE OF CELL IN BEAM
  • 15.
    CELL SIZE ANDVOLTAGE INTENSITY
  • 16.
    Electronics convert opticsignals to voltage pulse
  • 18.
    SIDE SCATTER • Scatterat larger angle. (~90o from beam axis). • Because of granularity and complexity inside the cells. • Focused to the side scatter detection system and detected by separate detectors. • Detection is same as forward scatter detectors.
  • 19.
    FLUORESCENT DETECTION • Fluorochromemolecules have Excitation (Accept light at given wavelength) and Emission (Re-emit light at higher wavelength) processes. 41 3 1. Light is absorbed by fluorochrome and electrons become excited. 2. Excited electrons migrate from Resting (Ground) state to Excited state. 3. Within 10 nanoseconds it releases some of absorbed energy as heat and fall lower to more stable level. 4. Electron steadily moves back to Ground state by releasing energy as Fluoroscence.STOKES SHIFT 2
  • 20.
    FLUORESCENT DETECTION • AbsorbedEnergy (Eexcitation) > Released Energy (Eemission) • Stokes Shift = Eexcitation - Eemission. • Value of Stokes Shift determines quality of fluorochrome. • Higher the value of Stokes Shift easier the detection. • Fluorescent Probes directly target the interested cells so readily chosen for flowcytometric analysis. • More parameters can be detected one at a time if more fluorochromes are used. • Generally Tandem Dyes are used for detection. •E.g. Alexa Fluor 488, Phycoerythrin, APC Cy 7.
  • 21.
    FLUORESCENT DETECTION FLUORESCENT LABELLED Ab CELL •Fluorophores are attached to the cell surface or inside the cell. • Fluorescent signals are emitted when cell passes through laser light (635 nm, 488 nm). • Signals are collected by various mirrors and filters and detected by Photodiodes or PMTs.
  • 24.
    Quantifying FACS Data FACSdata collected by the computer can be displayed in two different ways
  • 25.
    Here we seea different way to display the same data. • The X-axis plots the intensity of green fluorescence while the Y-axis plots the intensity of red fluorescence. • The individual black dots represent individual cells and we are not supposed to count the dots but just look at the relative density of dots in each quadrant. Cont.….
  • 28.
    Magnetic Activated CellsSorting(MACS)  A flexible, fast and simple magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers.  Cells stained sequentially with biotinylated antibodies, fluorochrome-conjugated avidin, and superparamagnetic biotinylated-microparticles (about 100 nm diameter) are separated on high gradient magnetic (HGM) columns.  More than 109 cells can be processed in about 15 min.  The simultaneous tagging of cells with fluorochromes and very small, invisible magnetic beads makes this system an ideal complement to flow cytometry.  Light scatter and fluorescent parameters of the cells are not changed by the bound particles.  Magnetically separated cells can be analyzed by fluorescence microscopy or flow cytometry or sorted by fluorescence- activated cell sorting without further treatment.  Magnetic tagging and separation does not affect cell viability and proliferation.
  • 29.
    Cont.…  Bound largeparticles have disadvantages.  Magnetic microparticles (diameterparticl(e ( 0.5 pm) can have a variety of superior characteristics compared to larger particles.  Their major disadvantage is the small magnetic moment, resulting in long separation times in magnetic fields.  To overcome this disadvantage, Molday and Molday have suggested a combination of small superparamagnetic microparticles and high gradient magnetic (HGM) fields with Yields Relatively High Purity Cell Populations.
  • 30.
    MACSŽ Technology  3Components Involved  MACS MicroBeads  MACS Column  MACS Separator  MACS MicroBeads  50 nm(superparamagnetic particles) in size  Biodegradable  Conjugated to monoclonal antibodies  MACS Column  Generates strong magnetic field
  • 31.
    Direct Labeling  Advantages Fast  Yields High Purity Cell Populations  Disadvantages  Binding of antibody to surface marker may activate cell  Some cell populations may require multiple markers to be identified Indirect Labeling • Useful in cases with dim surface marker expression • Allows for amplification of signal • Secondary antibody recognizes • Biotin • FITC • Fc portion of primary antibody • Streptavidin instead of secondary antibody • Recognizes biotin on primary antibody Bead Monoclonal Ab Labeling Of Cells With Beads
  • 32.
  • 33.
  • 34.
    Magnetic Separation High Gradient MagneticField Trapped Labeled Cells Cells Of Interest
  • 35.
    Positive selection Negative selection,cellDepletion  Positive selection: desired cell population is magnetically labeled and isolated as the retained cell fraction.  Negative selection: Depletion of undesired cells. Non-target cells are magnetically labeled and depleted from the cell mixture. The flow through contains desired cell fraction.  Useful In Following Cases  Removal of Unwanted Cells  No specific antibody is available for target cell  Binding of antibody to target cell results in activations.
  • 36.
  • 37.
  • 38.
  • 39.
    Applications of FACSand MACS Utility in assisted reproduction Isolation Of CD4+ Cells Characterization of multidrug resistance (MDR) cells Multiplex protein assay cytometric bead array Cell Cycle Analysis Analysis of Intracellular Ion Concentration Differentiating cancerous cells Chromosome analysis and sorting Measuring cell membrane permeability Stem cell identification
  • 40.
    Magnetic activated cellsorting (MACS): Utility in assisted reproduction  Assisted reproductive techniques (ART) have now been extensively incorporated in the management of infertile couples.  Conventional semen analysis .  Sperm apoptosis has been heavily linked to failures in reproductive techniques. One of the earliest changes shown by apoptotic spermatozoa is externalization of phosphatidyl serine.  Magnetic activated cell sorting (MACS) is a novel sperm preparation technique that separates apoptotic and non-apoptotic spermatozoa based on the expression of phosphatidylserine.  By separating the apoptotic spermatozoa it has also improved the success rates of assisted reproduction techniques.
  • 41.
    How does itwork???  MACS technology uses annexin V-conjugated superparamagnetic microbeads (50 nm) to separate nonapoptotic spermatozoa from those with deteriorated plasma membranes and externalization of PS.  Depending on Ca2+, PS has a high affinity for annexin V, which is 35-36 kDa phospholipid binding protein.  Annexin-V does not have the ability to pass the intact sperm membrane, so the annexin-V binding to spermatozoa characterizes disturbed integrity of the sperm membrane.  Thus annexin enables the identification of cells with altered membrane integrity Based on annexin binding and subsequent magnetic separation 2 fractions are obtained.  annexin-negative(unlabeled-intact membrane; non-apoptotic) and annexin positive (labeled- altered membrane; apoptotic).
  • 42.
    Isolation Of CD4+Cells Use CD4(L3T4) Microbeads.
  • 43.
    Charactrization of multidrugresistance (MDR) cells • The multidrug resistance (MDR) phenotype is associated with the overexpression of members of the ATP-binding cassette family of proteins. These MDR transporters are expressed at the plasma membrane. • The multidrug resistance protein 1 (MRP1), P-glycoprotein, and the breast cancer resistance protein are each present in a subcellular compartments and perinuclear region. • Fluorescence-activated cell sorting detection of the three most studied transporters: P-glycoprotein, multidrug resistance-associated proteins, and breast cancer resistance protein.
  • 44.
    Cont.…  Detection ofP-glycoprotein using the antibody MRK16  Detection of MDR1 Expression using antibody CD243
  • 45.
    Multiplex protein assaycytometric bead array  A multiplex assay is a type of assay that simultaneously measures multiple analytes (dozens or more) in a single run/cycle of the assay.  Multiplex assays are widely used in functional genomics experiments that endeavor to detect or to assay the state of all biomolecules of a given class (e.g., mRNAs, proteins) within a biological sample.  Multiplexing offers several distinct advantages over single-plex assays
  • 46.
    Cytometric Bead Array(CBA) assays  In bead-based assays, immunodetection occurs not on the flat surface of a membrane or microtiter plate but on micron-sized spheres.  Each bead contains a unique blend of fluorophores that acts as a signature and is associated with a single analyte—bead identifier 1 corresponds to IL-2, identifier 2 to IL-4 and so on.  Multiplexing is accomplished by combining different bead sets (with associated capture antibodies) into one master mix and incubating that mix with each sample in a microtiter plate.  Several multiplexed bead systems are available. Cytometric Bead Array (CBA) assays, which can be multiplexed to 30 analytes.
  • 47.
    Cell Cycle Analysis One of the most common uses of FACS is to analyze the cell cycle of mammalian cells.  FACS can measure the deoxyribonucleic acid (DNA) content of individual cells at a rate of several thousand cells per second and thus conveniently reveals the distribution of cells through the cell cycle.  G2/M-phase cells have twice the amount of DNA as G1/G0-phase cells, and S- phase cells contain varying amounts of DNA between that found in G1 and G2 cells.  Furthermore, each subpopulation can be quantified
  • 48.
    Analysis of IntracellularIon Concentration  An alteration in intracellular calcium concentration is one of the most common second messenger responses now known in mammalian cells.  The vast majority of changes in intracellular calcium is extremely rapid and occurs within a nanosecond timescale.  The recent development of a number of new fluorescent probes makes it possible to measure the concentrations of various intracellular free ions in single living cells.  Among these ions are calcium, magnesium, sodium, potassium, and hydrogen (pH).  Using the dyes Indo-1 AM, Fluo-3, and Fura Red AM to measure intracellular calcium concentration.
  • 50.
    Differentiating cancerous cells Some of the determinants of tumor response seem to be expressed at the cellular level in terms of degree of tumor cell differentiation.  Quantitative cytology in the form of flow cytometry has greatly advanced the objective elucidation of tumor cell heterogeneity by using probes that discriminate tumor and normal cells and assess differentiative as well as proliferative tumor cell properties.  Among phenotypic tumor cell markers, surface membrane antigens have been extensively studied in lymphoid and myeloid neoplasms by the use of hybridoma-generated monoclonal antibodies, which have recently also found in vitro and in vivo therapeutic application.
  • 51.
    Cont.….  FACS isused for the identification and effective isolation of CSCs .  Similar to hematopoietic stem cells or stem cells from other tissues, measurements of specific cluster of differentiation (CD) surface markers and stem cell-specific metabolic activities have been used for the characterization of CSCs.  The list of CD markers found to be associated with CSCs is extensive and includes many markers used for the identification of nonmalignant hematopoietic or mesenchymal stem cells and other special markers like the B-cell marker CD20 for melanomas .
  • 54.
    Chromosome analysis andsorting  Conventional karyotyping involves the cytogenetic analysis of a metaphase spread.  FACS from monodispersed suspension of chromosomes.  A good chromosome preparation is derived from a semi-confluent cell culture which is an exponential phase of growth.  DNA dyes - chromomycin A3 which stains CG-rich regions of DNA and Hoechst 33258 which stains AT-rich regions.  The chromosomes then separate according to their DNA content (size) and base pair composition.
  • 55.
    Measuring cell membranepermeability  A reliable and rapid test to detect cytotoxic chemicals which affect cell membranes.  Fluorescein diacetate freely penetrates intact cells.  On the other hand, ethidium bromide is known to be excluded from the intact cell.  The combination of both fluorochromes results in counter-staining: intact cells fluoresce green (cytoplasm) and membrane-damaged cells fluoresce red (nucleus and RNA).
  • 56.
    Cont.…. Human ovarian carcinomacell line labelled with FDA and PI(propidium iodide). The viable cells are fluorescein +ve, PI -ve and the dead cell fluorescein -ve, PI +ve. Green: live cells. Red: dead cells. Some of the blue cells are probably apoptotic.
  • 57.
    Some other Applications: Counting T cell and B cell  Protein expression and localization.  Transgenic products in vivo (particularly the green fluorescent protein (GFP)  Immunophenotyping by related fluorescent cell surface antigens (Cluster of differentiation (CD) markers), intracellular antigens (various cytokines, secondary mediators, etc.), nuclear antigens.  Enzymatic activity, pH, apoptosis.  Measurement of DNA degradation, mitochondrial membrane potential, permeability changes.
  • 58.
  • 59.
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  • 60.
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