Flow Cytometry

A. Aims:

In this lab session, you will learn how the flow cytometer works, and you will be
provided with an overview of its applications, the type of data generated and its
analysis.

B. Learning Objectives:

1. Identifying the different components of the flow cytometer

2. Understanding how the flow cytometer works
3. Describing applications of flow cytometry
4. Analyzing histograms obtained from different flow cytometry experiments

C. Introduction:

The flow cytometer is an advanced machine that is being increasingly used in the
medical field and biological research for various applications. The name refers to the
ability of this machine to measure (meter) the characteristics of single cells (cyto),
suspended in a flowing stream of isotonic fluid (flow).

Figure 1: The flow cytometer facility available at the CRSL, Physics Department,
AUB.

1. Properties of a Flow Cytometer:

The flow cytometer is characterized by its ability to measure characteristics (physical

and/or chemical) of one cell at a time, and to process thousands of cells per second (a
flow cytometer can discriminate cells at a speed up to 30,000 cells per second, far
outpacing a hematologist who can count 200 cells in less than a minute under the
microscope). This machine is accurate, and it allows for a diverse range of studies
involving cell counting, sorting and biomarker detection, which increases its
significance in research and accounts for its various uses in the medical and
pharmaceutical fields.

2. Major Components of a Flow Cytometer:

The flow cytometer has four main components:

 Fluidics: Delivers the cells in a single file to the interrogation point, where the
laser intersects with the sample. This is accomplished by hydrodynamic focusing,
where a sample containing randomly distributed cells is injected and limited to an
outer stream of sheath fluid at higher pressure, thus creating a stream of single
cells. The flow rate is controlled through adjusting the sample pressure.
 Optics: Consists of the laser beam, optical filters, lenses, mirrors, prisms and other
optical components. Its role in general is to generate, focus and collect light
signals.
 Electronics: Converts the optical signals into proportional electronic signals and
digitizes them, in order to perform computer analysis of the collected data.
 Computer: Displays, analyzes, stores and edits data collected.

3. Principle of the Flow Cytometer:

A focused beam of laser hits each cell individually as cells pass through it in a
continuous flow of a very fine stream of the suspension. The light is then scattered
forward and sideways by the cells. This information is gathered by detectors,
converted into proportional electronic signals that are further digitized and then
displayed by the computer for analysis. Forward scattered light correlates with the
size of the cell, whereas the intensity of side scattered light indicates nuclear shape
and granularity of the cell. Further properties such as cell surface molecules or
intracellular constituents can be studied as well. This is accomplished through the use
of fluorescent dyes (or fluorophores) that can bind cellular components, such as DNA,
or that are conjugated to antibodies specific to cell membrane or intracellular
molecules. Each fluorophore has a characteristic peak excitation and emission
wavelength. The intensity of fluorescent light emitted is then used to study the cellular
marker in question. The use of multiple fluorophores with similar excitation
wavelengths and different emission wavelengths, or the use of blue laser which is
capable of exciting several fluorophores with different excitation wavelengths, makes
it possible to study several cellular properties simultaneously.

4. Uses of the Flow Cytometer:

 Fluorescence-Activated Cell Sorting (FACS):

The flow cytometer allows the separation of different cells in a suspension based on
their size (forward scattered light), granularity (side scattered light) or the presence of
cellular markers (fluorescence) in case a fluorescent tag is used. It works as such:

 A cell suspension containing cells labeled with a fluorescent dye is directed into a
thin stream of fluid, so that all the cells pass in single file. The dye is coupled to a
 monoclonal antibody and binds to those cells coated with the antigen for which
the antibody is specific.
 This stream emerges from a nozzle vibrating at some 40,000 cycles per second,
which breaks the stream into 40,000 discrete droplets each second (some of these
may contain a cell).
 A 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 the signals from the two detectors meet either of the criteria set for fluorescence
and size, an electrical charge (positive or negative) is given to the stream.
 The droplets retain this charge as they pass between a pair of charged metal plates:
o Positively charged droplets are attracted to the negatively charged plate
and pass into a specific container.
o Negatively charged droplets are attracted to the positively charged plate
and pass into another container.
o Uncharged droplets (those that contain no cells or cells that fail to meet the
desired criteria of fluorescence and size) pass straight into a third container
and are later discarded.

This apparatus can sort as many as 300,000 cells per minute. In addition, the cells are
not damaged by the process; rather, because the machine can be set to ignore droplets
containing dead cells, the percent viability of the sorted cells can be higher than that
in the original suspension.
Figure 2: Separation of cells using a fluorescent-activated cell sorting (FACS) via the
flow cytometer.

 Cell Counting:

For medical purposes, it is sometimes essential to count the number of a specific sub-
population of cells. For example, it is of utmost importance to determine the amount
of B and T lymphocytes in blood for clinical diagnosis. Other studies could be done
using the flow cytometer for medical diagnostic purposes. For example, counting T-
cell subsets in human blood requires fluorescent monoclonal antibodies directed
against each of the CD4 and the CD8 molecules. CD4+ T cells (or T helper cells)
provide help to B cells and are responsible for several cell-mediated immune
responses. CD8+ T cells are cytotoxic T lymphocytes (CTLs). This test is important
because the prevalence of CD4+ over CD8+ T cells is typical of healthy humans,
whereas in AIDS patients, this ratio becomes reversed and the CD4+ subset may
eventually disappear.
Figure 3: Cell counting by using fluorescent stains specific for CD4+ (left) and CD8+
(right) T cells. Such a spectrum, where CD4+ T cells predominate over CD8+ T cells,
is the case seen in normal, healthy humans. The x-axis indicates fluorescence
intensity, while the y-axis indicates the number of cells for the specific intensities (not
shown here).

 Cell Cycle Analysis:

In many pharmacological studies, there is a need to test the effect of a chemical or

drug on both normal cells and cells of the disease or case under study. One of the
many experiments done uses flow cytometry to analyze the effect of added chemicals
on the cell cycle of treated cells and the distribution of cells in the different stages of
the cycle. This, in turn, allows the determination of the effect of such chemicals on the
cell in general and identifies the stage of the cell cycle at which this effect appears.
Such studies are performed using the fluorescent DNA-binding dye propidium iodide
(PI). The amount of fluorescence gives a direct insight into the amount of DNA in the
cell, implying the stage in which the cells are, thereby allowing a histogram
separation of the cells in the population into the different cell cycle stages.
Figure 4: Histogram of an asynchronous cycling cell population. Propidium iodide is
a commonly used dye to quantitatively assess DNA content. Cells in G0 or G1 phases
of the cell cycle possess a normal DNA content (2n), whereas those in G2 phase and
mitosis (M phase) contain exactly twice this amount (4n). As DNA is synthesized
during S phase, cells are found with a DNA content ranging between 2n and 4n. Cells
falling in the sub-G1 phase would be considered to be apoptotic. The x-axis indicates
fluorescence intensity, while the y-axis indicates the number of cells for the specific
intensities.
(http://www.icms.qmul.ac.uk/flowcytometry/uses/cellcycleanalysis/propidiumiodide/i
ndex.html)

 Assessment of Gap Junction Functionality:

In some biological studies and research projects, it is essential to assess the

functionality of gap junctions, as it provides valuable information regarding the
interactions of cells in homogeneous or heterogeneous cultures. This can be
performed using the flow cytometer by a technique called "calcein dye transfer
assay".

In the results shown below (Figure 5), human mammary epithelial carcinoma cell line
(MCF-7) is tested for functional gap junctions between cells of the same type (i.e.
MCF-7 with MCF-7) using calcein dye transfer assay. This experiment involves the
following:

 Two sets of cells are cultured: one set of cells is kept normally growing, while the
other set is incubated with calcein dye. Calcein dye is in the esterified form and is
non-fluorescent and membrane permeable; however, when it enters the cell it is
cleaved by intracellular esterases into a fluorescent, membrane-impermeable form,
which can only be transferred between cells through gap junctions.
 After incubation with the dye (at a specific concentration that depends on the cell
type used) for a period of 1-2 hours (also cell type dependent), the media
containing the dye is removed, and fresh media is added to the cells and kept for
15 minutes at 37 ºC. This allows the removal of any non-esterified dye that is still
present.
 Afterwards, the cells labeled with calcein dye are removed, counted and plated at
specific density over the set of cells that were kept unlabeled.
 This incubation is done for 30 minutes to 2 hours at 37 ºC.
 Afterwards, the unattached, labeled cells are removed with the media.
 The adherent cells, which represent the previously unlabeled cells, are removed,
fixed with formaldehyde and analyzed by flow cytometry.
 The presence of intermediate levels of dye in these previously-unlabeled cells is
indicative of functional gap junctions between the labeled and unlabeled cells.

M1

M2

Figure 5: Calcein dye transfer assay. The x-axis indicates the intensity of
fluorescence, while the y-axis indicates the number of cells for the specific intensities.
The peak at the extreme left of the plot refers to absence of fluorescence due to
unlabeled cells, while the peak at the extreme right refers to maximum fluorescence
due to labeling of the cells with calcein dye. Any intermediate peak would imply
intermediate fluorescence in the cells, which could only be due to transfer of dye from
labeled to unlabeled cells via gap junctions. In this graph, the black plot refers to
unlabeled (negative control) MCF-7 cells, whereas the green plot refers to the
unlabeled cells after culturing with labeled MCF-7 cells for a period of 2 hours. The
shift in fluorescence (FL1) to the left shows that there has been dye transfer to the
unlabeled cells.