FACS and MACS with their applications in biological research.
Application of FACS and MACS in
Dr. Aparna Chaudhari
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
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
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.
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
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
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.
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
• The fluidic system is used to transport particles from a random
three-dimensional sample suspension to an orderly stream of
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
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
Longpass filters permit longer wavelength transmission, while shortpass filters allow shorter
Bandpass filters only allow a selected wavelength band of interest to be transmitted while
blocking unwanted wavelengths.
Silicon photodiodes and photomultiplier tubes (PMTs).
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
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
• Low angle scattering (upto 20o
from beam of axis).
• Detector converts intensity of
light into voltage.
• Blocking bar is placed in front of
• 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.
ABSENCE OF CELL IN BEAM
PRESENCE OF CELL IN BEAM
Electronics convert optic signals to
• Scatter at larger angle. (~90o from
• Because of granularity and
complexity inside the cells.
• Focused to the side scatter
detection system and detected by
• Detection is same as forward scatter
• Fluorochrome molecules have
Excitation (Accept light at given wavelength) and
Emission (Re-emit light at higher wavelength) processes.
1. Light is absorbed by
fluorochrome and electrons
2. Excited electrons migrate from
Resting (Ground) state to
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
• 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
• 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.
• 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
• 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.
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.
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
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.
3 Components Involved
50 nm(superparamagnetic particles) in size
Conjugated to monoclonal antibodies
Generates strong magnetic field
Yields High Purity Cell Populations
Binding of antibody to surface marker may activate cell
Some cell populations may require multiple markers to be identified
• Useful in cases with dim surface marker expression
• Allows for amplification of signal
• Secondary antibody recognizes
• Fc portion of primary antibody
• Streptavidin instead of secondary antibody
• Recognizes biotin on primary antibody
Labeling Of Cells With Beads
Positive selection Negative
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
No specific antibody is
available for target cell
Binding of antibody to
target cell results in
Applications of FACS and MACS
Utility in assisted reproduction
Isolation Of CD4+ Cells
Characterization of multidrug
resistance (MDR) cells
Multiplex protein assay cytometric
Cell Cycle Analysis
Analysis of Intracellular Ion
Differentiating cancerous cells
Chromosome analysis and sorting
Measuring cell membrane
Stem cell identification
Magnetic activated cell sorting (MACS): Utility in
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
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
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
• Fluorescence-activated cell sorting detection of the three most studied
transporters: P-glycoprotein, multidrug resistance-associated proteins, and breast
cancer resistance protein.
Detection of P-glycoprotein using the antibody MRK16
Detection of MDR1 Expression using antibody CD243
Multiplex protein assay cytometric bead
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
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
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
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
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.
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
DNA dyes - chromomycin A3 which stains CG-rich regions of DNA and Hoechst 33258 which stains AT-rich
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).
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
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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.