Encapsulation A sample of cells, either derived from an
in vitro cell culture or from an
in vivo test subject is dispersed into individual cells and suspended in molten low-melting-point
agarose at 37 °C. This mono-suspension is cast on a
microscope slide. A glass
cover slip is held at an angle and the mono-suspension is applied to the point of contact between the coverslip and the slide. As the coverslip is lowered onto the slide the molten agarose spreads to form a thin layer. The agarose is gelled at 4 °C and the
coverslip removed. The agarose forms a matrix of
carbohydrate fibres that encapsulate the cells, anchoring them in place. The agarose is considered to be
osmotic-neutral, therefore
solutions can penetrate the gel and affect the cells without cells shifting position. In an
in vitro study the cells would be exposed to a test agent – typically
UV light,
ionising radiation, or a
genotoxic chemical – to induce DNA damage in the encapsulated cells. For
calibration,
hydrogen peroxide is usually used to provide a standardized level of DNA damage.
Lysis The slides are then immersed in a solution that cause the cells to
lyse. The lysis solution often used in the comet assay consists of a highly concentrated aqueous
salt (often, common
table salt can be used) and a
detergent (such as
Triton X-100 or
sarcosinate). The
pH of the lysis solution can be adjusted (usually between neutral and
alkaline pH) depending upon the type of damage the researcher is investigating. The aqueous salt disrupts
proteins and their bonding patterns within the cell as well as disrupting the
RNA content of the cell. The detergent dissolves the
cellular membranes. Through the action of the lysis solution the cells are destroyed. All proteins, RNA, membranes and
cytoplasmic and
nucleoplasmic constituents are disrupted and diffuse into the agarose matrix. Only the
DNA of the cell remains, and unravels to fill the cavity in the agarose that the whole cell formerly filled. This structure is called nucleoid (a general term for a structure in which DNA is concentrated).
Electrophoresis After lysis of the cells (typically 1 to 2 hours at 4 °C) the slides are washed in
distilled water to remove all salts and immersed in a second solution – an
electrophoresis solution. Again this solution can have its pH adjusted depending upon the type of damage that is being investigated. The slides are left for ~20 minutes in the electrophoresis solution prior to an
electric field being applied. In alkaline conditions the
DNA double helix is
denatured and the nucleoid becomes single stranded. An electric field is applied (typically 1
V/
cm) for ~20 minutes. The slides are then neutralised to pH 7, stained with a
DNA-specific fluorescent stain and analysed using a
microscope with an attached CCD (
charge-coupled device – essentially a
digital camera) that is connected to a computer with
image analysis software. ==Background==