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 particlessuch as cells, suspended in a stream of fluid.

It allows simultaneous multiparametric analysis of the physical and/or chemical characteristicsof up to thousands of particles per second.

FCM is used in the diagnosis of health disorders, and has many applications in both research andclinical practice.

It is used To Count T cell and B cell

An impedance-based flow cytometry device was patented in 1953; the first fluorescence-basedflow 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 separationof 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 morefluorescent detectors.

Each suspended particle –from 0.2 to 150 micrometers- passing through the beam scatters the light, and fluorescentchemicals found in the particle or attached to it may be excited, emitting light at a longer wavelength than the lightsource.

This combination of scattered and fluorescent light is recorded by the detectors and, by analyzing changes in brightnessat 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, theamount 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 activelyisolate 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 (normalincidence) filters.

Longpass filters permit longer wavelength transmission, while shortpass filters allow shorterwavelength transmission.

Bandpass filters only allow a selected wavelength band of interest to be transmitted whileblocking 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 AbCELL

• 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 cellsaccording to specific cell surface markers.

Cells stained sequentially with biotinylated antibodies, fluorochrome-conjugated avidin, andsuperparamagnetic biotinylated-microparticles (about 100 nm diameter) are separated on highgradient 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 beadsmakes 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 orsorted 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.

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

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 ofmembers of the ATP-binding cassette family of proteins. These MDR transportersare expressed at the plasma membrane.

• The multidrug resistance protein 1 (MRP1), P-glycoprotein, and the breastcancer resistance protein are each present in a subcellular compartments andperinuclear region.

• Fluorescence-activated cell sorting detection of the three most studiedtransporters: P-glycoprotein, multidrug resistance-associated proteins, and breastcancer 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 membraneor microtiter plate but on micron-sized spheres.

Each bead contains a unique blend of fluorophores that acts as a signature and isassociated with a single analyte—bead identifier 1 corresponds to IL-2, identifier 2to IL-4 and so on.

Multiplexing is accomplished by combining different bead sets (with associatedcapture antibodies) into one master mix and incubating that mix with each sample in amicrotiter 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 ofdegree of tumor cell differentiation.

Quantitative cytology in the form of flow cytometry has greatly advanced the objective elucidationof tumor cell heterogeneity by using probes that discriminate tumor and normal cells and assessdifferentiative as well as proliferative tumor cell properties.

Among phenotypic tumor cell markers, surface membrane antigens have been extensively studied inlymphoid and myeloid neoplasms by the use of hybridoma-generated monoclonal antibodies, whichhave 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 phaseof growth.

DNA dyes - chromomycin A3 which stains CG-rich regions of DNA and Hoechst 33258 which stains AT-richregions.

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 ofdifferentiation (CD) markers), intracellular antigens (various cytokines, secondarymediators, etc.), nuclear antigens.

Enzymatic activity, pH, apoptosis.

Measurement of DNA degradation, mitochondrial membrane potential, permeabilitychanges.

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Magnetic activated cell sorting (MACS): Utility in assisted reproduction* Kartikeya Makker, Ashok Agarwal† &

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and Gynecology and Women’s Health Institute, Cleveland Clinic, Cleveland, Ohio, 44195 USA.