Monography, Flow Cytometry analysis

Instituto Superior TécnicoMestrado Bioengenharia e NanossistemasProjecto Integrado II

Flow CytometryAnd applicationsAntónio Filipe Sousa, MBioNano

Flow cytometry and applications

António Filipe Sousa

Contents1. Introduction......................................................................................................................2

1.1. Flow Cytometry – Definition........................................................................................3

1.2. Principles of Flow Cytometry.......................................................................................3

2. Aplications of flow cytometry in stem cell analysis.............................................................6

3. Stem cell Phenotype characterization using flow cytometry...............................................7

3.1 –Human Mesenchymal Stem Cells (hMSC).......................................................................7

3.1.1. Protocol......................................................................................................................8

3.1.2. Results and discussion...............................................................................................8

3.2. – Mouse Embryonic Stem Cells (mESC)..........................................................................10

3.2.1. Protocol....................................................................................................................11

3.2.2. Results and discussion.............................................................................................11

4. Study of transfection efficiency of hMSC using flow cytometry.........................................12

4.1. Transfection of hMSC using lipofectamine and corresponding flow cytometry analysis...............................................................................................................................................12

4.1.1. Protocol....................................................................................................................13

4.1.2. Results and Discussion.............................................................................................13

5. Study of human embryonic kidney 293 (HEK293) cells transfection efficiency using flow cytometry analysis...............................................................................................................13

5.2.1. Protocol....................................................................................................................15

5.2.2. Results and Discussion.............................................................................................15

6. Conclusions......................................................................................................................17

7. Acknowledgment.............................................................................................................17

8. References.......................................................................................................................18

2

Fig. 1 - Schematic view of the main components of the Cytometer. 1 – Fluidic system; 2 – Lasers; 3 – Optics; 4 – Detectors; 5 – Electronics and computer system; 6 – interrogation point.



1. Introduction

1.1. Flow Cytometry – Definition

Flow cytometry is a powerful technique for the analysis of multiple parameters of

individual cells within heterogeneous populations. Flow cytometers are used in a wide range of

applications, such as immunophenotyping, cell counting and reporter gene (e.g. green

fluorescence protein (GFP)) expression analysis [1]. This simultaneous parametric

model analysis of the physical and/or chemical characteristics is obtained by passing

thousands of cells per second through a laser beam and capturing the light of each cell as it

passes through it. The data gathered can be analyzed statistically by flow cytometer software

to report cellular characteristics such as complexity, size, phenotype or viability, as well to

purify populations of interest with Fluorescence-activated cell sorting (FACS) . This technology

has a high number of applications, including molecular biology, immunology and in medicine

(e.g. transplantation, tumor immunology and chemotherapy, genetics, and sperm sorting for

sex pre-selection). The use of fluorescence tagged antibodies is useful in the field of molecular

biology as they bind to specific antigens giving unique information about the cells being

studied in the cytometer.

1.2. Principles of Flow Cytometry

The Flow Cytometer is

composed of several

components; figure 1 shows a

schematic representation of

the interior of this equipment.

The main components are: the

fluidic system, which presents

samples to the interrogation

point and takes away the waste

after that point; the lasers

which produce a beam of light

3

Fig. 3 – a: Laser beam passing through the particle and consequent FS (1) and SS (2); b: obscuration bar (1) and light sensor (2).

Fig.2 – Schematic representation of the hydrodynamic focusing in flow cytometer.



of a single wavelength and is directed onto a hydrodynamic focusing stream of fluid; the optic

lenses that gather and direct the light; a number of detectors which are aimed at the point

where the stream passes through the light beam: one in line with the light beam and several

perpendicular to it; and finally the electronics and the peripheral computer system,

responsible for the conversion of the electrical signal into digital

data and to perform the necessary analyses. The interrogation

point is the heart of the system. This is where the laser and the

sample intersect and the optics collects the resulting scatter and

fluorescence.

For a good data collection, the particles or cells in study

must pass through the laser beam one at a time. This is obtained

by injecting the sample stream containing the cells into a

flowing stream of sheath fluid or saline solution, as represented

in figure 2. The sample stream becomes compressed and so

narrow that roughly one cell passes through the channel at a time – this is called

hydrodynamic focusing. Cytometers are able to detect particles between 1 and 15 microns in

diameter.

Once the laser hits the cell, light will be refracted in all directions. Two different types

of light scattering can be considered: Forward scattering (FS) and side scattering (SS) (figure

3a). As the light strikes the cells, it is scattered in the forward direction to the sensor - (FS). The

magnitude of the FS light is proportional to the size of the cell, and this data can be used to

quantify that parameter. To quantify this light

most flow cytometers have a blocking bar

(obscuration bar) (figure 3b). Thus, once the

cells passes through the laser beam, light is

scattered around this component and

collected by the sensor. This prevents any

intense laser light from reaching the sensor.

Small cells produce small amount of FS and big cells produce large amount of FS, so the

magnitude pulse recorded for each cell is proportional to the cell size. Plotting a histogram of

this information will put smaller cells in the left and larger cells in the right; In another way,

once the laser beam hits the cells, light will be scattered in all directions, or in different words,

in larger angles – SS. This SS at higher angles is caused by granularity and structural complexity

inside the cells, it is focused through a lenses system and is collected by a separate sensor,

4

a b

Fig.4 – a1: plots from FS and SS; a2: two dimensional dot or scattered plot, here it’s possible to see that the dots correlate to the peaks of the FS and SS plots; b: 2D scatter plot of blood, representing lymphocytes (low internal complexity), monocytes (medium sized cells and slightly more complex) and granulocytes (high internal complexity).



usually located at 90 degrees from the laser’s path. Like it happens in the FS, the signals

collected from the SS light detector can be plotted on one dimension histogram.

By studying only the forward scatter light, it is not possible to obtain a complete

information about a population. For example, what appears to be a single population through

FS analysis, can in fact be multiple populations that can be only separated by SS analysis and in

a two dimensional plot of the resulting data. This is done through the use of two-dimensional

dot or scatter plots. The peaks from the forward and side-scatter histograms correlate with the

colored dots in the scatter plot, figure 4a.

As an example, we can

take the scatter plot of

a typical peripheral

blood cell run, and see

the results in a side

scatter plot, using

forward and side

scatter data (figure

4b). Here it is possible

to observe

lymphocytes, which

are small cells with

low internal complexity, monocytes which are medium-sized cells with slightly more internal

complexity, and neutrophils and other granulocytes which are large cells that have a high

internal complexity.

When using flow cytometry it is possible to study cellular characteristics by labeling

them specific antibodies with linked to fluorescent molecules. These antibodies will bind to the

cell surface, or even to molecules inside the cells. In the same way as described above, when

laser light of the right wavelength strikes the fluorescent molecule, a fluorescent signal is

emitted and detected by the sensor. In a solution with

cells, some of them will be brighter than others, after

passing through the laser beam, the fluorescent light will

be translated into a voltage pulse proportional to the

amount of fluorescence emitted, and then this

information can be presented graphically.

5

Fig. 5 – Forward scatter threshold.

Fig.6 - Pluripotent, embryonic stem cells can be isolated from the inner mass of the blastocyst. These stem cells can become any tissue in the body, excluding the placenta. Only the morula's cells are totipotent, able to become all tissues and the placenta [2].



While collecting flow cytometry data, the use of a threshold (or discriminator or

trigger) may be important. Indeed, since all the single

particles that pass through the laser are counted,

without the definition of a threshold, the dominant information that would appear would be

concerning about minor particles such as platelets and debris. The purpose of creating a

threshold is to set that a certain forward scatter pulse size must be exceeded for the

instrument to collect the data. In figure 5, the area in the left of the red line represents the

small cells and debris that are excluded from the analysis by the threshold. In this way, the

major data that is presented by the flow cytometer corresponds to the cells of interest,

although the small particles are still passing through the instrument, but being ignored.

2. Aplications of flow cytometry in stem cell analysis

First of all, it is important to explain the

concept of stem cells so we can understand how

flow cytometry can be used as an important tool,

for the study of stem cells. Stem cells are

undifferentiated cells that have the ability of self-

renewing themselves and that are capable of

originating multiple cell lineages or more

restricted progenitor populations, which in turn

generate precursors and then fully mature cells.

Stem cells may be found in the embryo and in

adult tissues, contributing to tissue homeostasis

by regenerating tissue after injury. The capacity

to differentiate into specialized cell types defines

the potency of the stem cells. Thus, stem cells

can be totipotent, having the capacity to specialize into all cell types, including the

extraembrionic membrane and tissues; pluripotent, having the capacity to differentiate into

cells from all the embryonic germ layers (endoderm, mesoderm and ectoderm); and

multipotent that only differentiate into a limited range of cell types (figure 6) [2]. Other cells,

named as progenitor cells, can divide a limited number of times before facing a change in their

potency or undergoing differentiation [3].

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In the adult body, it is likely that each tissue has a pool of stem cells that maintain their

multipotency under strict growth control and can be mobilized to intervene in injury scenarios

[4]. So the study of the molecular, cellular and development biology of embryonic and adult

stem cells is a very powerful approach to understand the organization and function of complex

tissues and organs [5]. Flow cytometry can be used for this purpose.

Stem cells can be characterized namely by the expression of several transcription

factors and cell surface proteins. As exposed above (section 1.2), the identification and

quantification of expression of cellular antigens with fluorochrome-labeled monoclonal

antibodies (“immunophenotyping”) is one of the most important applications of the flow

cytometer. For flow cytometry analysis it is necessary to prepare single cell suspensions and to

make them react with one or several immunoflurescent antibodies that will attach to the

antigen whose expression is being analyzed.

For this work, two different scenarios were explored and will be presented. First, two

different examples will be presented to illustrate how flow cytometry can be used for the

characterization of the phenotype of both adult and embryonic stem cells. After that, it will

also be illustrated how flow cytometry can be used to quantify the percentage of transfected

stem cells and other types of mammalian cells after using respectively two different

transfection techniques: lipofection and microporation.

3. Stem cell Phenotype characterization using flow cytometry

3.1 –Human Mesenchymal Stem Cells (hMSC)

Multipotent mesenchymal stem cells (MSC) are non-haematopoietic stromal cells that

are capable of differentiating into distinctive end-stage cell types, such as bone, cartilage,

muscle, bone marrow stroma, tendon/ligament, fat, dermis, and other connective tissues as

diagrammed in Figure 7. MSC can secrete a wide spectrum of bioactive immunoregulatory

molecules which play an important role in tissue regeneration [6]. Though not immortal, MSC

have the ability to expand many-fold in culture, whilst retaining their growth and multi-lineage

potential [7]. MSC of human origin (hMSC) are subject of intense and important study as they

have useful clinical applications [8]. So, an important part of this study is to investigate

whether or not hMSC maintain their characteristic phenotype after in vitro culture.

7

Fig. 7 - The Mesengenic

Process diagram originated

in the late 1980s as a

hypothesis based on what

was known about

mesenchymal progenitors in

embryos. The format was

designed to mirror the

lineage pathways of

hematopoiesis with the

bone lineage (Owen, 1985)

on the left reflecting the

state of knowledge, while

the lineages at the right

were largely unstudied. The

original diagram appeared

first in Caplan (1989).



Presently, a set of standards is well established to define hMSC for both laboratory-

based scientific investigations and for pre-clinical studies. First, MSC must be plastic-adherent

when maintained in standard culture conditions using tissue culture flasks. Second, ≥95% of

the MSC population must express cell surface markers CD105, CD90 and CD73. Third, the cells

must be able to differentiate to osteoblasts, adipocytes and chondroblasts under standard in

vitro differentiating conditions [9].

CD105, CD90 and CD73 (CD stands for complement of differentiation) are antigen

molecules expressed in the membrane of hMSC. At the molecular level, an antigen is

characterized by its ability to be “bound” at the antigen-binding site of an antibody.

In this work, flow cytometry was performed for hMSC immunophenotype characterization

using antibodies that were labeled with a fluorescent dye, Phycoerythrin (PE). By virtue of its

huge absorption coefficienct and almost perfect quantum efficiency PE it is one of the

brightest dyes used today. It emits at about 570 nm, which corresponds to a green light

detected in the flow cytometer [10].

3.1.1. Protocol

The protocol of the extracellular staining of cells used for flow cytometry is presented

in annex I

3.1.2. Results and discussion

8

Fig. 8 – Flow cytometry results for the expression of the

extracellular markers (CD105, CD90 and CD73) in hMSC. From top

to down CD 73, CD 90 and CD105. The histograms on the left show

the results obtained with the medium DMEM supplemented with

10% FBS, and on the right, with low serum commercial

MesenPROTM.



In this work, flow cytometry was used to evaluate the phenotype of hMSC after in vitro

expansion in the presence of two different culture media: DMEM supplemented with 10% FBS

(Fetal bovine serum) and the low

serum commercial MesenPROTM. hMSC

were cultured in the presence of these

two culture media and afterwards the

percentage of hMSC expressing CD105,

CD90 and CD73 was evaluated by flow

cytometry. There is a number of

parameters that needs to be inputted

to the flow cytometer, such us the

voltage of the laser beam and the

acquisition rate. After doing so, and

using the dot-plot graphic

corresponding to the physical

characteristics of the cells (as

explained in section 1.2), a gate of

viable cells was selected, and their

fluorescence was then quantified. As

previously explained, it is possible to

draw this gate in the flow cytometer

user interface so we can focus our

study in the desired cells. Cell debris or other cells that are outside the gate will be excluded

from the analysis. An important aspect of flow cytometry analysis is the necessary use of a

negative control run. For this purpose, isotypes of the monoclonal antibodies labeled with PE

were used. In this way, it was possible to detect unspecific binding. In figure 8, the unspecific

binding was represented by the first pick (1) of fluorescence in each graphic. After running the

negative control, as depicted in (1), the fluorescence of the sample can also be measured.

These values correspond to the second peak (2) of each graphic in figure 8.

As can be observed in figure 8, flow cytometer results shows that, under both culture

conditions (DMEM supplemented with 10% FBS and MesenPROTM), more than 95% of hMSC

9



express these characteristic markers. Thus, both culture media that are being used for the

culture of the hMSC allow the correct phenotype to be expressed.

3.2. – Mouse Embryonic Stem Cells (mESC)

Embryonic stem (ES) cells are pluripotent cells that have the capacity of almost

unlimited self-renewing, and they are able to differentiate into multiple cell types of the three

embryonic germ layers: ectoderm, mesoderm and endoderm [11]. To date, most of the

investigation has taken place using mouse embryonic stem cells (mES) or human embryonic

stem (hES) cells. Both have the same essential characteristics, but they require very different

culture conditions in order to maintain an undifferentiated state. The most important

consideration is that, without optimal culture conditions, embryonic stem cells will rapidly

differentiate [5].

Undifferentiated ES cells are evaluated as good material for applications in

regenerative medicine, pharmacological and toxicological studies. So it is important to know

and understand the factors affecting ES cell expansion and/or controlled differentiation in

order to obtain a high number of cells for application in such areas [12].

In this work, mouse ES cells will be used to illustrate the application of flow cytometry

for characterization of the phenotype of pluripotent stem cells. During in vitro expansion of

mES cells it is crucial to maintain the undifferentiated state of these cells during long periods of

time. This can be obtained by culturing the cells in serum-containing medium supplemented

with leukemia inhibitory factor (LIF) [13]. However the use of serum imposes some limitations

as it is a potential factor of pathogenic transmission. So, in this context, flow cytometry was

used to evaluate the possibility of using serum-free culture medium to successfully support

mES cells proliferation and maintenance of features during long periods of time [14].

Such as for the hMSC described in the section above, the expression of several typical

mES cell markers, such as the intracellular markers Oct-4 and Nanog and the extracellular

marker (SSEA-1) were evaluated by flow cytometry. Oct-4 and Nanog are both transcription

factors critically involved in the self-renewal of undifferentiated ES cells. Oct-4 expression must

be closely regulated; too much or too little will actually cause differentiation of the cells, and is

associated with an undifferentiated phenotype and tumors [15]. Overexpression of Nanog in

mES cells causes them to self-renew in the absence of leukemia inhibitory factor. In the 10



absence of Nanog, mES cells differentiate into visceral/parietal endoderm [16]. The antigen

SSEA-1 (stage-specific embryonic antigen-1) is expressed at the morula stage in embryos. It is

considered to function as a cell-cell interaction ligand in the compaction process. SSEA-1 is

expressed also in undifferentiated F9 teratocarcinoma cells, which cease to express it after

induction of differentiation [17].

3.2.1. Protocol

The protocol for the intracellular and extracellular staining of cells used for flow

cytometry is presented in annex I

3.2.2. Results and discussion

mES cells can be expanded in serum-free conditions through activation of STAT-3

signaling by supplementation of exogenous LIF and induction of differentiation (ID) proteins by

supplementation of bone morphogenic proteins (BMPs) [18]. For this work, the influence of

initial cell density under serum-free conditions was studied in the expansion of mES cells. For

that purpose, mES cells were plated at four different initial cell densities (10 4, 5x104, 105 and

5x105 cells/mL).

After expansion of mES cells, phenotypic

analysis by flow cytometry revealed that,

independently of the initial cell density,

expanded cells expressed high levels of

pluripotency markers, such as the cell surface

11

Fig. 9 – Pluripotent Mouse ES cells express high levels of transcription factors Oct-4 and Nanog, and cell surface marker SSEA-1 following expansion in serum-free medium, as assessed by flow cytometry analysis. Cells incubated only with secondary antibody, in the case of Oct-4 and Nanog, or with γ1-FITC isotype, for SSEA-1, were used as negative controls.

Fig. 10 - Schematic representation of various transfection technologies and how they neutralize the negatively charged DNA.



marker SSEA-1 and the transcription factors Oct-4 and Nanog (figure 9). Indeed, almost 95% of

the cells expressed Oct-4 and Nanog for all initial cell densities used. Concerning the SSEA-1

marker, the percentage of positive cells obtained was lower and between 70 and 94%.

Overall, these results show that culture under serum-free conditions is able to

maintain mouse ES cells pluripotency since proper signals are exogenously supplemented to

the culture medium.

4. Study of transfection efficiency of hMSC using flow cytometry

Transfection is the process of introducing genetic material into eukaryotic cells using

non-viral methods [19]. Normally this technique involves the opening of transient pores in cell

membrane, to allow the entering of nucleic acids material by using various chemical, lipid or

physical methods. Thus, this gene transfer

technology is a powerful tool to study gene

function and protein expression in the context of a

cell [20]. The transfected DNA and RNA are

negatively charged molecules, so the critical

problem of transfection is how to introduce these

molecules into cells that also have negatively

charged membranes (figure 10). This problem can

be solved by using chemicals, like calcium

phosphate and DEAE-dextran, or cationic lipid-

based reagents that coat the DNA, neutralizing or even creating an overall positive charge in

this molecule. Other physical methods, like microinjection or electroporation, simply punch

the DNA through the membrane introducing it directly into the cytoplasm. The following

section describes and discusses the use of lipid carriers for transfection of hMSC and the

corresponding analysis of transfection efficiency through flow cytometry.

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4.1. Transfection of hMSC using lipofectamine and corresponding flow cytometry

analysis

Lipofectamine is a common commercial transfection reagent. It is used to introduce,

which means to transfect, siRNA or plasmid DNA into in vitro cell cultures. Lipofectamine

treatment alters the cellular plasma membrane, allowing nucleic acids to cross into the

cytoplasm.

The mechanism of cationic lipid-mediated transfection originates from the basic

structure of cationic lipids: a positively charged head group and one or two hydrocarbon

chains. The positive surface charge of the liposomes mediates the interaction of the nucleic

acid with the cell membrane, allowing for fusion of the liposome/nucleic acid (“transfection

complex”) with the negatively charged cell membrane. The transfection complex is thought to

enter the cell through endocytosis. Once inside the cell, the complex must escape the

endosomal pathway and diffuse through the cytoplasm [21].

In this experiment, the purpose was the transfection of hMSC with the gene encoding

for the GFP (green fluorescent protein) aiming at the optimization of in vitro transfection

protocols for hMSC.

4.1.1. Protocol

The protocol used for the transfection of cells using lipofectamine is presented in

annex I

4.1.2. Results and Discussion

The results of this experiment are presented in annex II. Different quantities of plasmid

DNA and different quantities of lipofectamine were tested in order to optimize the transfection

protocol.

The results show that the percentage of hMSC expressing GFP varied between 14%

and 20%. Thus, although lipofection is considered a gentle method, being able to maintain high

levels of cell viability, in the case of these experiments, the transfection rate obtained was

relatively low.

13

Fig. 12 – Schematic representation of the cappilar used in microporation.



5. Study of human embryonic kidney 293 (HEK293) cells transfection

efficiency using flow cytometry analysis

Until now, this work was focused in application of flow cytometry for adult and

embryonic stem cell phenotype characterization and for evaluation of transfection efficiency

studies using stem cells. In this section, however, flow cytometry was used to determine the

transfection efficiency using a different mammalian cell line, the Human Embryonic Kidney

(HEK) 293 cells. HEK293 cells are a specific cell line originally derived from

human embryonic kydney cells grown in tissue culture. HEK293 cells are very easy to grow

and transfect and have been widely-used in cell biology research for many years. They are also

used by the biotechnology industry to produce therapeutic proteins and viruses for gene

therapy. Due to their importance in these fields, in this work the transfection of HEK293 cells

was optimized and the results were evaluated by using flow cytometry. For that purpose, the

yellow fluorescence protein (YFP) was cloned in a vector alongside with the nuclear antigen

EBNA (nuclear antigen) and transfected into HEK 293 cells. This EBNA sequence allows the

recombination of the plasmid DNA with the genetic material of the cells [22]. After

transfection, HEK293 cells will express fluorescence that will be proportional to the amount of

YFP present in the cytoplasm. Its excitation peak is

514nm and its emission peak is 527nm. YFP has

reduced chloride sensitivity, faster maturation, and

increased brightness (product of the extinction

coefficient and quantum yield).

In this particular case of HEK293,

electroporation was selected as the transfection

method. Electroporation is based in the significant

increase of the cell membrane electrical conductivity and permeability caused by

an external electrical field (Figure 11). Thus, by electroporation it is possible to

introduce genetic material into the cell, namely a piece of coding DNA, that will

originate a mutagenesis in a specific gene. The technique requires fine-tuning

and optimization of pulse duration and strength for each type of cell used. In

addition, electroporation often requires more cells than chemical methods

because of substantial cell death, and extensive optimization often is

14

Fig. 11- A diagram of the main components of an electroporator with cuvette loaded.



required to balance transfection efficiency and cell viability. Modern electroporation

instruments allow nucleic acid delivery to the nucleus and thus the successful transfer of DNA

and RNA to primary and stem cells. In addition, the use of capillary instead of the cuvettes

used in electroporation, allows microporation to be a more efficient technique. The gap size

between the two electrodes is maximized and the surface area of electrode can be minimized

compared to the cuvette type chamber, as shown in figure 12. By doing so, the transfection

efficiency and cell viability is dramatically increased.

Next, a schematic view of the experiment is presented next:

As exposed above, the process of microporation needs to be optimized for each cell

type. So, in this experiment, several conditions were tested for transfection of HEK293 cells.

Two different plasmids were tested: the pCEP4-YFP, with 11Kb of length, that was specifically

designed for these types of cells, and the pBGH-YFP, with 4Kb length. For each plasmid, several

voltages of microporation were used: 1000v, 1100v, 1200v, 1300v and 1400v. Finally the

percentage of microporated viable cells was quantified by flow cytometry.

5.2.1. Protocol

The protocol used for the electroporation of HEK293 cells is presented in annex I

15



5.2.2. Results and Discussion

As performed for the other analysis in the previous sections, after gating live cells in

the physical characteristics dot-plot, they are then analyzed concerning their fluorescence, as it

is shown in the histogram of figure 14. In all three histograms it is possible to see a peak inside

the M1 region representing the cells that are negative for the YFP expression. On the other

hand, the M2 region contains the cells that emit fluorescence. Indeed, the FL1-H axis is a

measure of the fluorescence emitted by the cells. The correspondent histogram statistics can

also be obtained using a specific software. With this we can see the percentage of counted

cells and also the percentace of gated cells in each region.

The first result presented corresponds to the negative control run (figure 14, a), a flow

cytometer analysis of HEK293 cells that were not transfected in the microporator. By

16

Fig. 14 – The first histogram, a) represents the negative control run with cells that weren’t transfected. The second graphic, b) quantifies the fluorescence of the cells transfected with pCEP plasmid, using 1400V. The last graphic, c) quantifies the fluorescence of the cells transfected with the pBGH plasmid, using 1400V. On the right it is the statistical analysis of the each corresponding histogram.

a

b

c



observation of the histogram in figure 14a (control run), it was possible to conclude that until a

level of fluorescence of 102, HEK293 cells are negative for expression of YFP (M1 region). In the

same way, only after 102 fluorescence, cells will be positive for the expression of YFP (region

M2). After running the negative control, HEK293 cells after microporation were analyzed by

flow cytometry to determine the percentage of transfected cells as can be seen in the M2

region (figure 14).

When comparing the results obtained with the two different plasmids tested, the best

results were achieved for the pCEP4-YFP plasmid, as it can be seen when comparing the

percentage of cells in M2 regions in figure 14b and c (45% for pCEP4-YFP and 40% for pBGH-

YFP). Normally, it is expected that a smaller plasmid (pBGH-YFP) will be more successfully

transfected into a cell, since the metabolic burden caused by this plasmid will be lower.

However, probably because pCEP4-YFP was specifically designed for HEK293 cells, a higher

amount of YFP expression was obtained.

After observing the results obtained, it is possible to conclude that the percentage of

viable transfection is relatively low. This may be attributed do to the fact cells do not support

the electric pulse during the transfection, and they die as a consequence. Another point is that

plasmid DNA may not be able to totally recombine with the genomic DNA of the cell, and thus

cells will not emit fluorescence In conclusion, and as it was said before, the microporation

technique requires optimization and study.

6. Conclusions

Flow cytometry is a versatile tool with enormous potential for the study of cells and

particles. It can be used to determine many morphologic, molecular, biophysical and functional

cellular characteristics and because of their unique analytical capabilities flow cytometry has

become an integral part of the biotechnology research. The main goal of this work was to show

some applications of flow cytometry in stem cell phenotype analysis and also as a tool for

quantifying the transfection efficiency of stem cells and other mammalian cell types. Flow

cytometry is also used on a daily basis in hospitals and commercial laboratories for analysis of

red cell, white cell, and platelet counts and to determine differential white cell counts. These

clinical applications can be used to observe cellular aberrancies and assist in the therapeutic

decisions as well as to help predicting clinical outcomes.

In a near future, with more studies at the molecular level and with further refinements

in the technology, opportunities to apply flow cytometry should be even more abundant.

17



7. Acknowledgment

Finally, I would like to thank the orientation of Doctor Margarida Diogo throughout all

this work and all the collaborators at the BERG/IBB research lab.

8. References

[1] Roger S. Riley, Michael Idowu. Principles and Applications of Flow Cytometry.

[2] Hans R. Schöler (2007). "The Potential of Stem Cells: An Inventory". in Nikolaus Knoepffler,

Dagmar Schipanski, and Stefan Lorenz Sorgner. Humanbiotechnology as Social Challenge.

Ashgate Publishing, Ltd. pp. 28.

[3] Ahmed, S. (2009). The Culture of Neural Stem Cells. Journal of Cellular Biochemistry 106:1–

6 (2009).

[4] Evans GS, Potten CS. 1991. Stem cells and the elixir of life. Bioessays 13:135–138.

[5] Sanberg P. 2007. Neural stem cells for Parkinson’s disease: To protect and repair. Proc Natl

Acad Sci USA 104:11869–11870.

[5] Chambers I, Colby D, Robertson M, et al. (2003). "Functional expression cloning of Nanog, a

pluripotency sustaining factor in embryonic stem cells". Cell 113 (5): 643–55.

[6] Arnold I. Caplan. Adult Mesenchymal Stem Cells for Tissue Engineering Versus Regenerative

Medicine. Journal of cellular physiology. J. Cell. Physiol. 213: 341–347, 2007.

[7] Mesenchymal Stem Cells: their Phenotype, Differentiation Capacity, Immunological

Features and Potential for Homing. Giselle Chamberlain, James Fox, Brian Ashton and Jim

Middleton. 10.1634/stemcells.2007-0197.

[8] Dominici, M., Blanc, K Le, Mueller, I., Slaper-Cortenbach, I., Marini, Fc, Krause, Ds, Deans, Rj

Keating, A., Prockop, Dj and Horwitz, Em (2006) 'Minimal criteria for defining multipotent

mesenchymal stromal cells. The International Society for Cellular Therapy position statement',

Cytotherapy, 8:4, 315 — 317.

[9] Dominici, M., Blanc, K Le, Mueller, I., Slaper-Cortenbach, I., Marini, Fc, Krause, Ds, Deans,

Rj, Keating, A., Prockop, Dj and Horwitz, Em (2006) 'Minimal criteria for defining multipotent

mesenchymal stromal cells. The International Society for Cellular Therapy position statement',

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Monography, Flow Cytometry analysis

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