2. • Introduction
• What is in-vivo/in-vitro test?
• Materials in assay
• Methods
In-vitro
In-vivo
• Limititaions
3. The single cell gel electrophoresis (SCGE), the
Comet Assay, is a fairly simple procedure by using a
micro gel and electrophoresis to detect DNA damage.
After image analysis the damage in DNA looks like a
comet, so that’s why it is known as Comet Assay. This
technique is further developed and introduced the use of
high alkaline conditions. This step increased the ability
of the assay to detect not only the double strand breaks
but also the single strand breaks.
4. In-vivo means In-vitro means
"within the living" in "within the glass" in
Latin, and refers to Latin, and refers to the
experimentation done in technique of performing
or on the living tissue of a given procedure in a
a whole, living organism. controlled environment
Animal testing and outside of a living
clinical trials are forms organism. Test tubes and
of in vivo research. Petri dishes are examples
of objects used in vitro
research.
5. • Test substance: Solid test substance should be dissolved in
appropriate solvents. The solvents should not produce toxic effects
at the dose levels used and should not be suspected of chemical
reactivity with the test substance.
• Positive control: Although there are numerous potential positive
control substances like ethyl nitrosourea, N-nitrosodimethylamine,
methyl methanesulfonate, ethyl methyl sulfonate.
• Negative control: Solvents for test substance preparation will be
used as negative controls like sodium carboximethylcellulose aqua
solution, corn oil.
• Test animals:
o Specie: Rats and mice are the usual species
o Sex: Males and females may be used in the comet assay
o Age: At the time of dosing, 7-9 weeks of age.
6. • Cells: L5178Y mouse lymphoma cells, TK6 human
lymphoblast cell, human peripheral lymphocytes.
• S9 mix: S9 fraction (The S9 fraction is the product of
a organ tissue, is most frequently used in assays that
measure the metabolism of drugs), Glucose-6-
phosphate, NADP, NaOH.
• Positive control: Methylmethane sulphonate,
Ethylmethane sulphonate.
• Agarose gel:
1. 1.0-1.5% standard agarose gel
2. 0.5% low-melting agarose gel
7. Treatment
DNA-Repair
Flow chart for both
methods
Single cell
suspension
Slide Image
preparation analysis
Alkaline Stain
lysis
DNA Alkaline
unwinding electrophoresis
8. 1. Experimental
design:
Compound Dose (mg/kg/day) No. of animals per
sex
Negative control 0 5
Positive control 200 5
Test compound Low(1/4 of high) 5
Test compound Medium(1/2 of high) 5
Test compound High 5
9. 2. Cell preparation:
Single cell should be done within 1 hour after
animal sacrifice. The liver, stomach and the bone
marrow are processed as cell culture. These samples
are stored on ice until slide preparation.
10. 1. Cell Culture:
Cells may be centrifuged. The cells are
transferred to a tissue culture flask. Flasks are placed
in a static CO incubator at 37 C. The cell grow in
2
suspension culture. Cell cultures must be used within
10 days from frozen stock.
11. 2.Experimental
design:
Components Composition Concentration Concentration
in S9-mix in culture
S9 4mL 40 vol% 2 vol%
G-6-P 2mLof 180 118 mM 5.90 mM
mg/mL sol.
NADP 2mL of 25 6.4 mM 0.32 mM
mg/mL sol.
KCl 2 mL of 150 30 mM 1.50 mM
mM sol.
12.
13. • Slide preparation:
Frosted end glass slides are dipped in 1% normal
melting point agarose and left to air dry. These
predipped slides can be stored in a airtight container for
approximately 1 month. Agar mix is dispensed onto the
predipped slide and covered with a clean cover slip.
• Cell Lysis:
Once prepared, the slides are immersed in
chilled lysing solution for atleast1 h or overnight in a
refrigerator in a light proof container.
14. • Unwinding and Electrophoresis:
The slides are randomly placed onto a dry, level
platform of a horizontal electrophoresis unit. Nucleoids
are left to relax and unwind at 2-10 C for 20 min
depending on the cell type. After alkali unwinding, the
slides are electrophoresed at 18 V. As DNA carrier a
net negative charge, the single strand fragments migrate
towards the anode. The temperature of the
electrophoresis solution at the start of unwinding, the
start of electrophoresis and the end of electrophoresis
should be recorded.
15. • Neutralization:
After it the slides are immersed
in the neutralization buffer for atleast 5 min. After
visualization the slides are stained.
• Comet visualization:
The comets visualized using a
florescence microscope linked to a computer via a CCD
camera, and measured using an image analysis system.
Heavily damaged cells exhibiting a microscopic image,
commonly referred to as hedgehog or ghost cells.
16.
17.
18. • Tail length:
It is defined as a measurement from the point of
greatest intensity within the comet head.
• Tail moment:
It is defined as the product of the tail length and
the fraction of total DNA present within the tail.
TM = tail length*100
head
• Tail intensity:
It is defined as the florescence detected by image
analysis in the tail, which is proportional to the amount
of DNA that has moved from the head region into the
comet tail.
19.
20. • It is simple, sensitive, versatile, speedy in use
• It tells us not just how much damage is present in cells, but
what form it takes.
• It become one of the standard methods for assessing DNA
damage, with applications in genototoxicity testing, human
biomonitoring and molecular epidemiology, ecotoxicology,
as well as fundamental research in DNA damage and repair.
• Limit of sensitivity is approximately 50 strand breaks per
diploid mammalian cell.
• It has ability to measure DNA single-strand breaks,
modifications to the method allow detection of DNA double-
strand breaks, cross-links, base damage and apoptotic nuclei.
21. http://www.springerprotocols.com/Abstract/doi/10.138
5/1-59259-179-5:163
http://www.nature.com/nprot/journal/v1/n1/full/nprot.2
006.5.html
http://wiki.answers.com/Q/What_is_the_Difference_bet
ween_in_vivo_and_in_vitro_testing#ixzz1vyhIeBxy
“The In Vitro and In Vivo Comet Assays” by Brian
Burlinson chapter 8
