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.

1. Application of FACS and 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 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.

5. INSTRUMENTATION

6. 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

7.  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.

8. 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

9. Light Collection
Laser beam Side scatter -
90⁰
Forward
scatter

10. 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.

11. 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.

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 scatters light in all directions when passes through the laser.
 Two types of scatters : Forward Scatter
Side Scatter

14. • 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

15. CELL SIZE AND VOLTAGE INTENSITY

16. Electronics convert optic signals to
voltage pulse

18. 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.

19. 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

20. 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.

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
FACS data collected by the computer can be displayed in two different ways

25. 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.….

28. 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.

29. 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.

30. MACS® 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

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. MACS separation unit

33. MiniMACS Separator
Magnet
MS Column

34. Magnetic Separation
High Gradient
Magnetic Field
Trapped Labeled
Cells
Cells Of
Interest

35. 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.

36. Automated MACS separation

37. Dynal versus MACS beads

38. Comparison FACS versus MACS

39. 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

40. 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.

41. 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).

42. Isolation Of CD4+ Cells
Use CD4(L3T4) Microbeads.

43. 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.

44. Cont.…
 Detection of P-glycoprotein using the antibody MRK16
 Detection of MDR1 Expression using antibody CD243

45. 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

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 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.

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 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 .

54. 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.

55. 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).

56. 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.

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.

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64. 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.
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 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.