• Introduction

• What is in-vivo/in-vitro test?

• Materials in assay

• Methods In-vitro In-vivo

• Limititaions

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.

In-vivo means "within the living" in Latin, and refers to experimentation done in or on the living tissue of a whole, living organism. Animal testing and clinical trials are forms of in vivo research. 

In-vitro means "within the glass" in Latin, and refers to the technique of performing a given procedure in a controlled environment outside of a living organism. Test tubes and Petri dishes are examples of objects used in vitro research.

• 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 specieso Sex: Males and females may be used in the comet assayo Age: At the time of dosing, 7-9 weeks of age.

• 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

Treatment

Single cell suspension

Slide preparation

Alkaline lysis

DNA unwinding

Image analysis

Alkaline electrophoresis

Stain

DNA-Repair

Flow chart for both methods

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

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.

1. Cell Culture:

Cells may be centrifuged. The cells are transferred to a tissue culture flask. Flasks are placed in a static CO2 incubator at 37 C. The cell grow in suspension culture. Cell cultures must be used within 10 days from frozen stock.

2.Experimental design:

Components Composition Concentration in S9-mix

Concentration in culture

S9 4mL 40 vol% 2 vol%

G-6-P 2mLof 180 mg/mL sol.

118 mM 5.90 mM

NADP 2mL of 25 mg/mL sol.

6.4 mM 0.32 mM

KCl 2 mL of 150 mM sol.

30 mM 1.50 mM

• 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.

• 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.

• 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.

• 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.

• 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.

http://www.springerprotocols.com/Abstract/doi/10.1385/1-59259-179-5:163

http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.5.html

http://wiki.answers.com/Q/What_is_the_Difference_between_in_vivo_and_in_vitro_testing#ixzz1vyhIeBxy

“The In Vitro and In Vivo Comet Assays” by Brian Burlinson chapter 8