Genomics and Applied Biology 2016, Vol.7, No.2, 1-7
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PBS readymade solution was used as solution. Standard blood culture was performed from the blood belonging to
the patient on whom SCE would be applied. 24 hours after culture, 100 µl BrdU solution (per tube) was added to
the culture. Harvest procedures that are performed in 72-hour blood culture were applied here. Preparations made
at the end of the harvest were stained with 2% giemsa for 10 minutes.
In preparations, SCE, stained with FPG (Fluorescence + Giemsa technique), the number of SCEs and their
distribution to chromosomes were examined using Olympus BX51 light microscope in the Applied Imaging
Karyotyping system by selecting the slides according to the appropriate metaphases. Terminal changes were
counted as one change whereas interstitial changes were counted as two changes. The total number of SCE
counted in a case (patient) was divided by the number of metaphases examined and thus a patient’s average
number of SCEs per metaphase was determined. An average of 50 metaphases were counted and photographed in
the patients and the controls.
1.3 Micronucleus test
In order to determine the number of MN, the method developed by Fenech (Fenech, 2000) and Kirsch-Volders et
al. (Kirsch-Volders et al., 2003) was modified and used. Blood samples taken from 22 women with breast cancer
who were about the same age were heparinized at a rate of 1/10 and then 6 drops (0.2 ml) of them were added to
the chromosome media in sterile conditions (Rencüzoğulları and Topaktaş, 1991). The cell culture was incubated
in the incubator for 68 hours at 37
1°C. At the end of the 68
th
hour, which is the duration of culture, culture tubes
were centrifuged for 15 minutes at 1 200 rpm, and the supernatant was removed. The 0.5-0.7 ml liquid that
remained at the bottom and contained the cells were mixed thoroughly and then the hypotonic solution kept in the
drying oven at 37°C was added to the tubes. The addition of this solution was implemented drop by drop and by
stirring. After 5 ml hypotonic solution was added to each tube, their caps were replaced and put in the incubator.
The cells were treated in hypotonic solution at 37°C for 5 minutes. At the end of the period, the tubes were
centrifuged at 1 200 rpm for 15 minutes and supernatant was removed. This time, cold fixative was added, 5 ml to
each tube, slowly and by stirring as in the case of addition of hypotonic solution. The fixative was prepared by
diluting 1 unit of acetic acid and 5 units of methyl alcohol mixture at a rate of 1/1 with 0.9 NaCl %. The cells that
were treated with the fixative at room temperature for 20 minutes were centrifuged at 1 200 rpm for 15 minutes,
supernatant was removed and again fixative was added to the tubes. This procedure was repeated twice and
fixatives different from the ones used in the first fixative were used (1 unit acetic acid and 5 units methyl alcohol).
At the end of the 3
rd
treatment with fixative, it was seen that the liquid remaining in the tube had become totally
clear. The liquid was centrifuged each time after addition of fixative and the supernatant was removed. After the
last centrifuge, when the supernatant was removed so that 0.5-0.7 ml liquid remained at the bottom, preparation
procedure was begun. The cells that accumulated at the bottom of the tube were mixed using a Pasteur pipette,
thereby homogenizing them. 4-5 drops were drawn into the Pasteur pipette from this cell suspension. Cell
suspensions were dripped, 1 drop in each area, on different areas on the sliding glass, which had been cleaned
before and kept in a fridge in pure water (4-5 drops on each sliding glass), and thus cells were enabled to spread
across the sliding glass. During the dripping of the cell suspension on sliding glasses, it was ensured that the drops
did not overlap. The preparations obtained in this way were kept at room temperature for 24 hours to dry. Then,
the preparations were colored with giemsa stain. The preparations stained with giemsa were examined under
Olympus BX51 light microscope in the Applied Imaging Karyotyping system. 1 000 cells that had not suffered
damage and had not been fused with other cells were counted. Micronuclei that had even edges, whose
dimensions were one third of the main nucleus and which showed the same coloring characteristics were
evaluated.
1.4 Binuclear cell
To obtain binucleated cells, cytochalasin B (6 mg/mL) was added to each culture 44 hours after starting cultures,
to inhibit cytokinesis. Cells were allowed to grow for another 28 hours, after which they were collected by
centrifugation, resuspended in a prewarmed hypotonic solution (0.075 mol/L KCl) for 15 minutes at 37°C, and
