This document summarizes an experiment that investigated the relationship between gamma radiation exposure and cellular senescence in human lymphocytes. 20 samples of lymphocytes were irradiated with doses from 0 to 4 Gray of gamma radiation. The cells' p16 proteins were then analyzed using fluorescent antibodies to identify senescent cells. The results showed a positive quadratic correlation between radiation dose and the occurrence of senescence, indicating more cells underwent senescence at higher doses. More trials are needed to establish a more accurate connection and reduce procedural errors.

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INVESTIGATING CELLULAR SENESCENCE FOLLOWING RADIATION EXPOSURE
Stephen Liu1 and Mihai Dumbrava2
1 Bayview Secondary School – 10077 Bayview Avenue, Richmond Hill, Ontario
2 Assumption College School – 257 Shellard Lane, Brantford, Ontario
Tutor: Adrienne Wan
Supervisor: Laura Paterson
Abstract
Cellularsenescence is the process in which cells lose their ability to proliferate,thereby limiting their lifespan [6].
One way senescence can be induced is through exposure to oncogenic (i.e.cancer-causing) stresses [10], which
includes high-energy radiation [11].Thisresearch project investigated experimentally the relationship between
gamma (γ) radiation and the process of cellular senescence. 20 samples of human lymphocytes, a type of small but
long-lived white blood cell, were irradiated with doses ranging from 0 to 4 Gray (Gy) and the cells’ p16 proteins
were analyzed using antibodieswith fluorescent probes [7].The experimental results revealed a positive quadratic
correlation between the amount of absorbed gamma radiation and the occurrence of senescence. It was also
indicated that more trials are necessary in the future to mitigate procedural errors and to establish a more
comprehensive and accurate connection between senescence and radiation.

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Figure 1 – Direct and indirect
actions of ionizing radiation.
1. Introduction
1.1 Background
CellularSenescence
Cellular senescence is a recently identified biological phenomenon that was first formally described less than
half a century ago [10]. It refers to the process in which cell growth and proliferation are irreversibly halted.
This mechanism has two main states and it is initiated in response to a range of DNA-damaging stimuli, both
intrinsic and extrinsic [11]. One state is replicative senescence,which occurs as a result of the progressive
shortening of DNA at the telomeres (i.e. the chromosomal ends)after each replication cycle. This pathway is
followed by cells that have a limited replicative potential. Conversely, stress-induced premature senescence
(SIPS) occurs in response to various oncogenic stressors,including radiation and toxic agents,and cannot be
attributed to the erosion of telomeres. Thus, by restricting the division of damaged cells, senescence is crucial in
preventing the transmission of mutations to daughtercells. This process differs from modes of cell death in that
senescent cells remain metabolically active, yet it also differs from quiescence in that these cells cannot be
stimulated to further proliferate.
Researchers recognize senescent cells by identifying the various phenotypes these cells express, including the
p16Ink4a (p16) protein and the senescence-associated secretory phenotype (SASP) [10]; these indicators are not
only essentialfor instigating and maintaining the senescence state,but also for communicating this state to the
surrounding microenvironment [11].
Gamma Radiation
This project examines SIPS through the utilization of gamma radiation, a form of
indirectly ionizing radiation [8]. This form of radiation deposits energy and
damages DNA in a two-step process:
1. Photons release charged particles (e.g. electrons or positrons)in a given
medium.
2. The released charged particles deposit energy through direct Coulomb
interactions with the orbital electrons of other atoms within the medium.
Afterwards, these energetic secondary electrons (as described in Steps 1 and 2
above) can damage DNA through two types of processes:direct and indirect
action (as shown in Fig. 1) [4]. In direct action, the secondary electrons interact
with the DNA to produce biological effects. Although this is a possible form of
interaction for all types of radiation, the dominant process for gamma rays is
indirect action [4]. This involves secondary electrons interacting with nearby
molecules (e.g. water) to produce highly reactive free radicals (i.e. molecules with
unpaired orbital electrons) that then damage the DNA [4].
Consequently,when individuals are exposed to large single doses of whole body irradiation, a wide range of
DNA anomalies and syndromes may manifest. Within the dose range of 4 to 7 Gy (1 Gray =1 Joule / Kilogram),
50% of human subjects will die if no form of medical intervention is provided; these values are known as the
lethal dose 50 (LD50) [5]. Moreover, the gradual accumulation of chromosomal damage can result in mutations
that lead to uncontrolled cell growth. At the same time, the same stimuli that can initiate and promote the
creation of cancer might also trigger a SIPS response; this mechanism can suppress the potentialof these
abnormal cells to grow into lethal tumors by stopping cell proliferation.
Fluorochromes
Identifying specific cellular components can be a challenge, so fluorochromes are frequently used to make
cellular properties more easily distinguishable. Fluorochromes are dyes that accept light energy at a given
wavelength and then re-emit it at a longer wavelength within a span of nanoseconds [3]. The applications of
these probes range from measuring enzyme activity to detecting cellular surface receptors [3]. This project
focuses mainly on identifying p16 proteins,a powerful tumour suppressorprotein and one of the critical
hallmarks shared by all senescent cells. As a result, p16 antibodies with attached Phycoerythrin (PE)
fluorochromes are used because they re-emit yellow light upon binding with p16 proteins, signalling the
occurrence of senescence.

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1.2 Purpose
The main purpose of this project is to observe how different doses of gamma radiation can affect cellular
processes,particularly the occurrence of senescence in human lymphocytes. This is done through analyzing the
cells’ p16 proteins using antibodies with fluorescent probes.By studying and comparing the percentage of
lymphocytes that senesce following various amounts of gamma emissions, the cellular consequences ofionizing
radiation can be betterunderstood.
1.3 Hypothesis
SIPS is induced by oncogenic stimuli including gamma radiation. On this premise, it was hypothesized that
more cells (i.e. a greater percentage) would undergo senescence following increased levels of radiation
exposure.
1.4 Application
Senescence has already been recognized for its various purposes in the human body,especially as a tumor
suppressor[6]. Not only do senescent tumourcells stop proliferating, but they also have a strong influence on
the division of neighbouring cells as demonstrated by experiments on model organisms [11]. Hence, by
investigating the occurrence of senescence after different doses ofradiation, the findings from this research can
enhance cancer treatments by identifying the level of radiation that maximizes the occurrence of this cellular
process.Currently, radiation therapy is a common procedure used to eliminate malignant tumors, but this
method is not always effective. If the treatment dose is too low, not all cancerous cells will be eliminated. If the
treatment dose is too high, the non-targeted healthy cells will be negatively affected. Thus,by exploiting this
cellular process,a better alternative treatment may be achieved.
In addition, collecting data on senescence may allow researchers to backtrack the amount of radiation received
by an individual in the case of an accidental exposure. This can be done through determining the proportion of
cells that have undergone senescence in a patient and comparing that to already-established senescence data to
determine an approximate range of radiation exposure (this will be discussed in further detail in Section 4.4
Future Research).
2. Materials, Experimental Method and Equipment
2.1 Materials
 Human Blood Samples
 Phosphate Buffered Saline (PBS)
 Ethanol
 Ficoll-Paque PLUS
 RPMI 1640
 Fetal Bovine Serum (FBS)
 Penicillin-Streptomycin
 Phytohemagglutinin (PHA)
 P16 Kit (BD Biosciences #556561)
 15 and 50 mL Centrifuge Tubes
 Sodium Heparin Vacutainers
 T25 Flasks
 Pipette Controllers
 5, 10 and 25 mL Pipettes
 Micropipettes
 Micropipette Tips
 Incubator
 Vortex Mixer
 Centrifuge
 Gamma Irradiator
 Imaging Flow Cytometer
2.2 Experimental Method and Specialized Equipment
Isolating Irradiated Lymphocytes
The initial steps in the procedure involved irradiating blood samples and isolating the layer of lymphocytes,
which is the blood component of interest in this project. Lymphocytes are a type of small white blood cell that
contribute to the body’s immune response,and they were chosen as the subjects for this research because they
are one of the most radiosensitive and reliable cell lines in the body [4]. Additionally, these cells are more
readily available compared to other sample types (e.g. tissue samples) and they have a lifespan of up to several

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years, providing a longer duration of time for relevant radiological research [12]. The following is the procedure
for isolating the irradiated lymphocytes:
1. Draw blood into sodiumheparin vacutainers
2. Mix blood by inversion
3. Irradiate blood (see Table 1)
4. Isolate lymphocytes using Ficoll-Paque PLUS
a. Add 3 mL of Ficoll-Paque PLUS to a 15 mL centrifuge tube
b. In a separate centrifuge tube, dilute 2.5 mL of blood sample 1:1 with PBS
c. Carefully layer blood solution (step b) on Ficoll-Paque PLUS
d. Centrifuge at 400 * g for 40 minutes at 20°C
e. Using a clean pipette, transfer lymphocyte layer to a new centrifuge tube. It is crucial not to
draw up any red blood cells
f. Add 8 mL of PBS to lymphocytes
g. Suspend cells by inversion
h. Centrifuge at 100 * g for 10 minutes at 20°C
i. Remove supernatant
j. Re-suspend lymphocytes in 7 mL of PBS
k. Repeat steps h and i
5. Create Complete RPMI Media by adding 41.9 mL of RPMI 1640, 7.5 mL of FBS and 0.5 mL of
Penicillin-Streptomycin into a 50 mL centrifuge tube
6. Transfer 10 mL of Complete RPMI Media into a T25 flask
7. Transfer lymphocyte suspension to flask
8. Add 100 µL of PHA to each flask
9. Incubate for 48 hours at 37°C, 5% CO2
10. Pour contents ofT25 flask into 15 mL centrifuge tube
11. Centrifuge for 5 minutes at 500 * g at 20°C
12. Discard supernatant into bleach waste
13. Re-suspend cell pellet
14. Wash cell suspension with 6 mL of PBS
15. Centrifuge for 5 minutes at 500 * g at 20°C
16. Discard supernatant into bleach waste
17. Re-suspend cell pellet
18. Repeat steps 13 to 16 for a second wash
Table 1 – Experimental Outline for Blood Sample Irradiation*
Absorbed γ Radiation Dose (Gy) Irradiation Time (s) Blood Volume (mL)
0 - 2.5
0.5 3.4 2.5
0.75 7.5 2.5
1 11.6 2.5
2 28.0 2.5
3 44.4 2.5
4 60.8 2.5
* The source of gamma radiation in the irradiator is Cobalt-60, which has a dose rate that gradually
decreased over theprogressive irradiation trials (from 61.1 mGy/s initially to 60.7 mGy/s by the
end); this discrepancy resulted from the decay of the radioactive isotopeover time and it was taken
into consideration when determining the irradiation times. Furthermore, the irradiation times were
specifically calculated to account for dead time in the gamma irradiator (i.e. the amount of time it
takes thesample in theirradiator to initially descend to theradioactive source and then resurface),
which typically contributes a radiation dose of 29.48 to 29.65 mGy.
Inserting p16 Antibodies
After the lymphocytes have been isolated, they must be prepared in such a way that p16 antibodies can attach to
intracellular p16 proteins within the senescent cells. This can be achieved by mixing the cells with ethanol,

5. 5
which fixates the cell and permeabilizes the cellular membrane, allowing the p16 antibodies to access all
subcellular compartments in search of the target protein [1]. The steps involved in this procedure include:
1. While vortexing, add 5 mL of cold 70% ethanoldropwise into cell suspension
2. Incubate at -20°C for at least 2 hours
3. Wash three times with 5 mL of PBS, centrifuge for 10 minutes at 500 * g at 20°C each time
4. Re-suspend cells and transfer 100 µL of cell suspension into each sample tube
5. Add 5 µL of properly diluted p16 antibody into tubes and mix gently
6. Incubate tubes at room temperature for 30 minutes in the dark
Flow Cytometry
Once all the cells have been treated with p16 antibodies,the final
step is to conduct the flow cytometric analysis. Flow cytometry is a
technology used to measure the multiple properties of individual
particles, usually cells, in a fluid as it passes through one ormany
lasers (as shown schematically in Fig. 2) [2]. Samples are
individually inserted into a flow cytometer, where the cells are
randomly distributed through a central channelthat is enclosed by a
sheath of fluid [9]. This layer of faster flowing fluid creates a large
drag force on the central solution,producing a single file of particles
[9]. This effect is called hydrodynamic focusing [9]. Afterwards,
each particle passes through at least one laser, and using an image-
capturing software called INSPIRE™ and a data analysis software
called IDEAS®, information about the particle’s properties can be
determined based upon the scattering of light or emission of
fluorescence (as shown in Figs. 3 and 4) [9]. The Channel (Ch) 01 in
Fig. 3 is a brightfield that shows the image of a cell under white
light, whereas each additional channelregisters and displays the
fluorescent signal from a cell after passing through different lasers;
cells with no dyes will only appear in Ch01. In Fig. 4, the yellow
emissions in Ch03 signify that p16 antibodies,along with the PE
dyes,have attached to this senescent cell and thus indicate the
presence of p16 proteins within the cell. Optimally, the fluorescence signal would only appear in Ch03, which is
the one intended to identify the light given off by the PE dyes.However, too much probe was added into this
cell sample and the resulting re-emissions were so bright that they were detected by every channel(this will be
discussed in further detail in Section 4.2 Error Analysis).
Figure 3 – Image of a non-senescent cell taken by the flow cytometer.
Figure 4 – Cytometric image of a senescent cell.
Furthermore, the INSPIRE™ software will store the data collected from cells and help to filter out the data
collected most non-cellular particles (e.g. dust). After the entire sample has passed through the flow cytometer,
all the images that were taken are compiled onto a single IDEAS® application file. At this point, these pictures
can be categorized through a process called gating, where images are graphed on a scatterplot based on visual
features (e.g. area) and cells of interest are marked off with a polygon (see Fig. 5). In the particular sample used
in Fig. 5, the first gate uses aspect ratio and area to identify cells from debris, the second gate uses circularity to
Figure 2 – Schematic diagram of a flow cytometer.

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identify regular cells from those with an abnormal morphology and the third gate uses the light intensity in
Channel 03 to identify senescent cells from non-senescent ones. Once all of the images have been properly
sorted,the percentage of senescent cells can be calculated using the equation:
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝑆𝑒𝑛𝑒𝑠𝑐𝑒𝑛𝑡 𝐶𝑒𝑙𝑙𝑠 =
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑆𝑒𝑛𝑒𝑠𝑐𝑒𝑛𝑡 𝐶𝑒𝑙𝑙𝑠
𝑇𝑜𝑡𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐶𝑒𝑙𝑙𝑠
× 100
Figure 5 – Gating procedure for a sample of cells.
3. Results
In this experimental research, 20 blood samples were irradiated with doses ofgamma radiation ranging from 0
to 4 Gy. After preparing each of them with p16 antibodies and conducting the flow cytometric analysis, the
measured results are as shown:
Table 2 – Experimental Senescence Data
Absorbed Radiation Dose Number of Senescent Cells Total Number of Cells Percentage of Senescent Cells
0 1 306 0.33%
0 0 367 0.00%
0 0 1098 0.00%
0 1 581 0.17%
0 0 182 0.00%
0 0 79 0.00%
0.5 1 178 0.56%
0.5 0 1386 0.00%
0.75 0 695 0.00%
0.75 0 307 0.00%
1 0 166 0.00%
1 1 158 0.63%
2 0 159 0.00%
2 1 825 0.12%
3 1 215 0.47%
3 0 141 0.00%
4 3 195 1.54%
4 0 283 0.00%
4 2 187 1.07%
4 1 40 2.50%
All
Single cellsSingle cellsSingle cellsSingle cellsSingle cellsSingle cells
900 1.5e30 600300 1.2e3
Area_M01
0.2
0.8
0
0.4
1
0.6
AspectRatio_M01
Area_M01, Aspect Ratio_M01
Population Count %Gated
All 842 100
Single cells 554 65.8
Single cells
CirclesCirclesCirclesCirclesCirclesCircles
30100 20
Circularity Ch01
30
0
20
10
CircularityCh01
Circularity Ch01, Circularity Ch01
Population Count %Gated
Single cells 554 100
Circles & Single cells 143 25.8
Circles
Senescent CellsSenescent CellsSenescent Cells
LiveLiveLive
Senescent CellsSenescent CellsSenescent Cells
LiveLiveLive
1e4 1e5-1e3 1e61e30
Intensity_MC_Ch03
1e5
1e3
1e6
0
1e4
-1e3
Intensity_MC_Ch03
Intensity_MC_Ch03, Intensity_MC_Ch03
Population Count %Gated
Circles & Single cells 143 100
Senescent Cells & Circles & Single cells 2 1.4
Live & Circles & Single cells 141 98.6

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Figure 7 – Mortality rates of rhesus monkeys after a
single total body dose to x-rays.
Figure 6 – The relationship between different absorbed doses of gamma radiation and the occurrence of cellular senescence.
A curve of best fit is also presented to approximate the general trend of the data.
3.1 Discussion
The data points in Table 2 showa positive correlation between the absorbed dose of gamma radiation and the
percentage of senescent cells within a sample. This is logical because as the amount of radiation increases, cells
that are affected are more likely to experience DNA damage that initiates the senescence response. Furthermore,
the relationship observed is one that is best approximated by a quadratic function, namely y = 0.0998x2
– 0.1052x.
This conclusion is in line with results in previously published literature about acute radiation syndrome (ARS),
which also describes the consequences of high levels of
radiation exposure within a short timeframe [4]. In a study
investigating the effects of radiation on a population of
rhesus monkeys, the relationship between the dose of
radiation and the percentage of monkeys killed is described
as a sigmoid function (Fig. 7) resembling the correlation in
Fig. 6 within the dose range of 0 to 4 Gy (1 Gy = 100 rads)
[4]. Between the absorbed dose of 0 and 2 Gy, there is close
to no mortalities, and the function increases towards 4 Gy in
a quadratic manner.
Although there is no established connection between the
consequences ofgamma radiation on cellular senescence and
cell death,it is possible that its effect on these two cellular
processes is analogous.As cells are exposed to more gamma
rays, the likelihood of the deposited energy inducing senescence and cell death could potentially remain
unchanged,thereby keeping the proportion of these cells similar (i.e. if the ratio of dead cells to senescent cells
is 10:1 at 6 Gy, it could remain 10:1 at 8 Gy).
3.2 Error Analysis
This project involves many laboratory procedures that leave room for human error. During the analysis of the
flow cytometric images, there were numerous images of debris and irregularly shaped particles that distorted the
cell samples. Much of this contamination may have resulted from improper aseptic techniques,such as dust
dropping into a sample from moving a glove over a test tube or particulates entering into a cell suspension after
using an accidentally contaminated pipette.
y = 0.0998x2 - 0.1052x
0
0.5
1
1.5
2
2.5
3
0 0.5 1 1.5 2 2.5 3 3.5 4
PercentageofSenescentCells(%)
γ Radiation Dose (Gy)
Effects of γ Radiation on Cellular Senescence

8. 8
Moreover, the senescence percentages may be less accurate due to the way the lymphocytes were prepared.
Firstly, the timespan of this project restricted the amount of time the lymphocytes could incubate and express
enough proteins to produce a detectable signal. Thus,there could have been senescent cells that were not
identified due to the lack of a senescence marker. In addition, the process of centrifuging the lymphocytes and
extracting the supernatant also contributed to this error. Although this step plays a crucial role in isolating the
lymphocytes, a small fraction of these cells would always be lost through each repetition, thereby widening the
margin of error.
Finally, statistical discrepancies may have also arisen from utilizing the IDEAS® analysis software. Similar to
how images of distorted particles were accidentally recorded, there is a possibility senescent cells were
overlooked by the program. Additionally, the process of gating the data to determine the cell populations often
included pieces of debris while excluding actual cells, resulting in a partially flawed representation.Lastly, the
senescence signalwas so intense that the morphology became unclear. This meant that senescent cells may have
been unaccounted forbecause they were indistinguishable by their image.
These types of errors could be reduced by being aware and vigilant during the sample preparation process,as
well as making adjustments to the procedure to better optimize the occurrence and detection of senescence.
Furthermore, the senescence data generated through this project is not very noteworthy,and more trials can be
conducted to mitigate the impact of these errors on the overall results of this research.
3.3 Conclusion
After irradiating lymphocytes at different levels of gamma radiation and plotting the doses against the
percentage of senescent cells,a positive quadratic correlation can be seen. This result is consistent with those in
publications regarding cell death,signifying that radiation exposure may affect the two cellular pathways in
similar ways.
However, errors during the experimental procedure and setup may have contributed to inaccuracies in the
results (as shown by outliers in Fig. 6). This can be corrected in the future by being more mindful of proper
aseptic techniques and adjusting the setup to optimize the occurrence and detection of cellular senescence.
Finally, tests with a larger range of radiation and more trials are necessary to produce results that can further
enhance the understanding ofthis phenomenon.
3.4 Future Research
Apart from continuing efforts aimed towards fully understanding cellular senescence,this research is part of a
larger project investigating how radiation exposure induces various types of cell death and growth arrest,
specifically apoptosis,necrosis and senescence. Different forms of radiation in various doses will have distinct
cellular impacts, and the goal of this study is to determine these unique markers and gather this information into
a single database for future reference. This can then be applied in situations such as radiological treatment,
where medical professionals may be able to extract blood samples from patients who suffered an accidental
radiation exposure and then extrapolate the initial dose based upon intracellular markers. This would allow for
more accurate diagnoses and more effective treatments.
4. Acknowledgements
Over these six weeks, I owe thanks to numerous individuals for making this incredible experience possible.
Firstly, I would like to thank the Deep River Science Academy, especially Margo Ingram, Shawna Kunkel and
Chantel Goodman, and the Atomic Energy Canada Limited’s Chalk River Labs for realizing this project and for
providing the world-class facilities. Next, I would like to thank Steve Pecoskie and Marilyne Stuart for their
constant technicalsupport and Samy El-Jaby for his valuable contributions towards my understanding ofthe
physics behind my work. Moreover, this project would not have been possible without the everlasting assistance
of my supervisorLaura Paterson, whose open attitude and positive energy always inspired me. In addition, I
would like to express many thanks to my wonderful tutor Adrienne Wan. Ever since day one, she has been an
effective teacher, enhancing my understanding ofthe project and pushing me to do my best. Finally, I would

9. 9
like to sincerely thank my exceptional project partner Mihai Dumbrava. He brought his keen and hard-working
mentality to work every day and it was a pleasure working with him.
5. References
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2014.
[3] AbD Serotec. "Fluorochromes and Light." Fluorochromes and Light. Bio-Rad Laboratories, 2014. Web. 01
Aug. 2014.
[4] Hall, Eric J., and Amato J. Giaccia. Radiobiology for the Radiologist.Philadelphia: Lippincott Williams &
Wilkins, 2006. Print.
[5] International Atomic Energy Agency. Radiation Biology:A Handbook for Teachers and Students.Vienna:
International Atomic Energy Agency,2010. IAEA. International Atomic Energy Agency, 2010. Web. 1
Aug. 2014.
[6] Kuilman, Thomas, Chrysiis Michaloglou, Wolter J. Mooi, and Daniel S. Peeper. "The Essence of
Senescence." Genes & Development. Cold Spring Harbor Laboratory Press, 2010. Web. 25 July 2014.
[7] Mayo Clinic Staff. "Lymphocytosis (high Lymphocyte Count)." Lymphocytosis Definition. Mayo
Foundation for Medical Education and Research, 30 July 2013. Web. 22 July 2014.
[8] Podgoršak, E. B. Radiation Physicsfor Medical Physicists. Berlin: Springer, 2006. Print.
[9] Rahman, Misha. "Introduction to Flow Cytometry." Introduction to Flow Cytometry. Bio-Rad Laboratories,
2014. Web. 24 July 2014.
[10] Rodier, Francis, and Judith Campisi. "Four Faces of Cellular Senescence." Four Faces of Cellular
Senescence.The Rockefeller University Press, 14 Feb. 2011. Web. 24 July 2014.
[11] Sabin, Rebecca J., and Rhona M. Anderson."Cellular Senescence - Its Role in Cancer and the Response to
Ionizing Radiation." Genome Integrity.BioMed Central, 11 Aug.2011. Web. 25 July 2014.
[12] Tough,D. F., and J. Sprent. "Lymphocyte life-span and memory." National Center for Biotechnology
Information. U.S. National Library of Medicine, May 1995. Web. 01 Aug. 2014.