2.5.2. Evaluation of the Micronuclei (MN) Appearance
The bone marrow cells were prepared following the method of Schmid (20)
with certain adjustments described by Bhilwade et al. (2004) (21). Each
animal’s femur was taken, and the excess tissues and muscles were
removed. The femoral marrow was flushed out using fetal bovine serum
(FBS) into a centrifuge tube and centrifuged at 1500 rpm for 10 min to
obtain cells pellet, which was thoroughly mixed, then smeared on coded
and cleaned frosted slides, air-dried and fixed with absolute methanol.
The slides were stained in May–Gruenwald for 5min, then with 10%
Giemsa for 10 min, followed by thorough washing with DW. The slides were
dried and examined under a light microscope. At least 1000 cells/animal
were screened for scoring the frequency of micronucleated polychromatic
erythrocytes (MNPCEs) (22).
2.6. Single-cell gel electrophoresis (SCGE) /Comet
assay
The comet assay was performed following the method described by Dhawanet al. (23) based on the original procedure developed by Singh et
al. (24). Concisely, the BM was flushed out with the chilled
Hanks’ Balanced Salt Solution (HBSS) buffer into a microcentrifuge tube;
then 5μl was mixed with 75μl of 0.5% low melting agarose solution
prepared in 0.9% normal saline and transferred onto frosted slides,
which were kept in lysis buffer (20 mM EDTA, 10% DMSO and 0.1% Triton
X-100) for 2 hours at 4 °C. Then the slides were removed from the lysis
buffer and placed on a horizontal electrophoresis gel box; then the
slides were kept in freshly prepared alkaline buffer (Electrophoresis
Buffer) with pH>13 for 20 min to unwind the DNA strands.
Electrophoresis was carried out for 30 min at 24 volts
(~0.74 V/cm), 300 milliamperes. The slides were gently
washed in a neutralising buffer for 5min, which was repeated twice to
remove the alkaline buffer and then dried. The slides were stained with
80μL 1X Ethidium Bromide, and a minimum of 50 cells/slide was captured
using a 40x objective on a fluorescent microscope. The comet images were
analysed using “Open Comet” digital imaging software. The percent (%)
DNA in Tail, which is the fraction of DNA in the comet tail divided by
the total amount of DNA associated with a cell multiplied by 100, was
measured to assess the extent of oxidative DNA damage.