Download LECTURE- 19: FLOW CYTOMETRY AND CELL SORTING

LECTURE- 19: FLOW CYTOMETRY AND CELL SORTING
Keywords: Cell sorting, DAPI, flowcytometry, propidium iodide.
1. Flow cytometry
Flow cytometry is a technique for counting and examining microscopic particles, such as cells
and chromosomes, by suspending them in a stream of fluid and passing them by an electronic
detection apparatus. It has established itself as a useful, quick and novel method to determine
efficiently and reproducibly the relative nuclear DNA content and ploidy level of a large number
of plant species. Basically, a flow cytometer is a fluorescence microscope which analyses
moving particles in a suspension. These particles are excited by a source of light usually a laser
and in turn emit an epifluorescence, which is filtered through a series of dichroic mirrors. The
inbuilt programme of the equipment converts these signals into a graph plotting the intensity of
the epifluorescence emitted against the count of cells emitting it at a given time. Thus, a flow
cytometer consists of fluidics, optics and electronics, as it measures cells in suspension that flow
in single-file through an illuminated volume where they scatter light and emit a fluorescence that
is collected, filtered and converted to digital values for storage on a computer. It allows
simultaneous multi - parametric analysis of the physical and/or chemical characteristics of up to
thousands of particles per second (Figure 19.1). For the fluorescence to be detected by the
photomultiplier, the cells have to be labelled with an appropriate fluorescent molecule whose
properties will change on binding to nucleic acids. The key feature of DNA probes is that they
are stoichiometric, whereby the number of molecules of the probe bound is equivalent to the
number of molecules of DNA found. Hence, when DAPI, for instance, binds to the AT base pairs
of DNA, the intensity of the fluorescence emitted will reflect the number of bounds and,
therefore, also the DNA content in such DAPI-labelled nuclei. Some fluorophores are listed in
Table 19.1.
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Figure 19.1: Schematic overview of a typical flow cytometer setup
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Table 19.1: Fluorophores used to label nucleic acids in flow cytometry
Fluorophore
Excitation maxima
(nm)
495, 342
Emission maximum
(nm)
639
Laser line
(nm)
488 A
372
456
UV A or K
493, 320
637
488 A
Acridine Orange
503
530
488 A
Mithramycin
445
569
457 A
Chromomycin A3
430
580
457 A
Hoechst 33342
395
450
UV A or K
Pyronin Y
545
565
530 K, 514 A
Thiazole Orange
509
533
488 A
Thioflavin T
422
487
457 A
Propidium Iodide
DAPI
Ethidium Bromide
One of the fundamentals of flow cytometry is the ability to measure the properties of individual
particles. When a sample in solution is injected into a flow cytometer, the particles are randomly
distributed in three-dimensional space. The sample must, therefore, be ordered into a stream of
single particles that can be interrogated by the machine’s detection system. This process is
managed by the fluidics system.
Essentially, the fluidics system consists of a central channel through which the sample is
injected, enclosed by an outer sheath that contains faster flowing fluid. As the sheath fluid
moves, it creates a massive drag effect on the narrowing central chamber. This alters the velocity
of the central fluid whose flow front becomes parabolic with greatest velocity at its centre and
zero velocity at the wall. The effect creates a single file of particles and is called hydrodynamic
focusing (Figure 19.2). Under optimal conditions, the fluid in the central chamber will not mix
with the sheath fluid.
Following hydrodynamic focusing, each particle passes through one or more beams of light.
Light scattering or fluorescence emission (if the particle is labelled with a fluorochrome)
provides information about the particle’s properties. The laser and the arc lamp are the most
commonly used light sources in flow cytometry. The stream of particles sequentially intersects
one or more laser beams placed orthogonal to the flow of fluid. The laser beams are placed such
that they only illuminate a single particle at any given time.
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Light that is scattered in the forward direction, typically up to 20⁰ offset from the axis of laser
beam, is collected by a lens known as the forward scatter channel (FSC) which roughly equates
to the particle’s size and can also be used to distinguish between cellular debris and living cells.
Light measured at a 90⁰ to the excitation line is called side scatter. The side scatter channel
(SSC) provides information about the granular content within a particle. Both FSC and SSC are
unique for every particle, and a combination of the two may be used to differentiate different cell
types in a heterogeneous sample.
Figure 19.2: Hydrodynamic focusing produces a single stream of particles
Signals are collected by an array of photo detectors. When light hits a photo detector, a small
current is produced. Its associated voltage has amplitude that is proportional to the total number
of light photons received by the detector. This voltage is then amplified by a series of amplifiers
into electrical signals which can be plotted graphically.
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Both fluorescence and scatter data are displayed in the form of univariate histogram or bivariate
scatter plots by specialized software. In the case of histograms, the x-axis will correspond to
either fluorescence or scatter intensity collected from a single photo-detector on either a linear or
logarithmic scale or the y-axis corresponds to the number of particles with the corresponding
light intensity. For example, if a cell is tagged with a fluorescent labelled antibody directed
towards a surface protein, the fluorescence will be directly proportional to the expression level of
this protein. By using multiple antibodies, one could assess the expression level of several
membrane bound and / or intracellular proteins of a single cell.
2. Cell Sorting
Because the flow cytometer display data from hundreds to thousands of cells that can highlights
different population of cells, it has been exploited to separate ultrapure populations of cells from
the original, heterogeneous sample and, thus, functions as a cell sorter. As the name implies, cell
sorters have the added benefit of being able to select individual particles of interest and divert
them from the fluid stream into a collection vessel. This is attained by introducing a slight
vibration on the nozzle to create small waves on the surface of the jet as it emerges from the
nozzle, causing it to break into regular droplets downstream of the point of laser intersection. If a
given particle is desired, an electric charge is applied to those drops containing the particles of
interest. Electrostatic charging occurs at a precise moment called the ‘break-off point’, which
describes the instant the droplet containing the particle of interest separates from the stream. The
particle-containing drops are deflected in an electric field and collected while the remaining
uncharged drops are disposed off. The amount of charge applied will affect the degree of
deflection of individual drops, and hence, multiple population of cell can be separated
simultaneously by using charge of different polarity and intensity (Figure 19.3). The speed of
cell sorting depends on several factors including particle size and the rate of droplet formation. A
typical nozzle is between 50-70 µM in diameter, and depending on the jet velocity from it, can
produce 30,000-100,000 droplets per second, which is ideal for accurate sorting.
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Figure 19.3: Electrostatic flow sorting
3. High Speed Cell sorting
Because each and every particle is analyzed individually, the performance of cell sorters is
intrinsically low. However, advances in fluidics, optics, computers and the electronics of cell
sorters have led to the development of instruments of high speed that can analyze and sort cells
at very high rates. It is important not to compromise sort quality and yield when designing a flow
cytometer capable of operating at high rates.
As particles are illuminated by one or more laser beams, signals that are detected by the photo –
detectors are sent to specialized circuits which perform analog to digital conversion, classify the
events and issue sort commands for those cells that fall within the specified sort windows.
Signals on the order of 1MHz can be easily processed by modern high level data acquisition
system and analog to digital convertors and therefore do not limit the overall sort process.
Modern high speed cytometers uses digital electronic circuitry which have little problem
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quantifying and classifying measurement parameters in the time it takes a cell to cross the laser
interrogation point. However, it is unlikely to sort events at a higher rate than that at which sort
drops are produced. Therefore, to attain the highest possible sort rate, the speed of drop
generation should approximate the speed of electronics.
4. Common Protocol - Sample preparation
Single cells must be suspended at a density of 105 – 107 cells/ml to prevent the narrow bores of
the flow cytometer and its tubing from clogging up. Phosphate buffered saline (PBS) is a
common suspension buffer for flow cytometry of non adherent cells. For analysis of cells from
solid tissues, the solid material must be disaggregated. In case of woody plant samples, generally
the woody plant buffer is used.
Preparation of plant tissue for flow - cytometric analysis
i.
About 20 mg of fresh sample explant like callus, leaves, seeds were taken in a
petriplate
ii.
Into the petriplate, 2 - 3 ml of the woody plant buffer was added and the sample was
chopped into fine pieces
iii.
After chopping, the suspension was filtered through a 30µm membrane
iv.
Into the filtered sample, 50µl/ml of RNase was added and was mixed well
v.
About 50µl/ml of propidium iodide was also added and the sample was mixed
vi.
The above steps are to be carried out at 4⁰C.
vii.
The sample prepared can then be introduced into the flow cytometer where it is
analyzed and evaluated accordingly.
5. Data Analysis
The ploidy level in the sample is determined by measuring the amount of nuclear DNA in the
plant cells. The flow cytometer automatically, rapidly and accurately makes this determination.
The distribution of the nuclear DNA content within a cell population is obtained by comparing
the number of cells in the different peaks or clusters. An internal standard can be used to run
together with nuclei suspensions of the genotypes analyzed and the position of the peak is used
to establish the ploidy level of such materials.
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Figure 4.3: Pure Diploid Sample, showing a peak in channel 100, as measured by the flow cytometer
Figure 4.4: Sample showing two peaks, one at 100 and another at 50, indicating a mixture of diploid
and haploid as detected by the flow cytometer
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6. Conclusion
Flow cytometric – based cell sorting is a well established technology that will continue to see
expansion of its uses in many areas of clinical and scientific research. Demands of the new
biology requires machine to function at higher speed and efficiency and capable of increasing
experimental complexity but at the same time should be robust and cheap. From a separation
perspective, cell sorting as an indispensable technology, where heterogeneous cells suspensions
can be purified into fractions containing a single cell type based upon virtually unlimited
combinations of user – defined parameters will lead to a more precise understanding of biology
and contribute to our increasingly detailed view of processes at the single cell level.
Study questions
1. What is the basic principle of flow cytometry?
2. Give some example of fluorophores which used for labelling nucleic acids in flow
cytometry?
3. Describe the hydrodynamic focusing in flow cytometry?
4. What is high speed cell sorting?
5. Describe the steps for plant sample proportion protocol for flow cytometry?
6. If a plant sample having tetra-ploid and peeks were coming at channel number 200 in
flow cytometry. At what channel number peeks will occur for following ploidy level of
same plant?
a) Haploid plant
b) Diploid plant
c) Hexaploid plant
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