Two smears were prepared for each treatment and air-dried prior to fixing in 90 % methanol at -20 °C for 20 min and staining with acridine orange (MP Biomedicals) for 2 min. After washing with phosphate buffer (Invitrogen, Carlsbad, CA, USA) twice for 3 min each, two slides per dose group were coded and scored blindly for MN in about 1000 reticulocytes (RETs) or polychromatic erythrocytes (PCEs) per slide at 1000x magnification under UV light using an Olympus BX50 fluorescent microscope (Southend-On-Sea, UK). We also determined the percentage of RETs or PCEs/normochromatic erythrocytes (NCEs) per 1000 cells, as any reduction in the number of PCEs or RETs is a sign of bone marrow toxicity.
Micronucleus Assay Protocol for Bone Marrow
Two smears were prepared for each treatment and air-dried prior to fixing in 90 % methanol at -20 °C for 20 min and staining with acridine orange (MP Biomedicals) for 2 min. After washing with phosphate buffer (Invitrogen, Carlsbad, CA, USA) twice for 3 min each, two slides per dose group were coded and scored blindly for MN in about 1000 reticulocytes (RETs) or polychromatic erythrocytes (PCEs) per slide at 1000x magnification under UV light using an Olympus BX50 fluorescent microscope (Southend-On-Sea, UK). We also determined the percentage of RETs or PCEs/normochromatic erythrocytes (NCEs) per 1000 cells, as any reduction in the number of PCEs or RETs is a sign of bone marrow toxicity.
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Corresponding Organization : The Women University Multan
Other organizations : National Institute for Biotechnology and Genetic Engineering
Variable analysis
- Last dose (time point)
- Micronuclei (MN) frequency in reticulocytes (RETs) or polychromatic erythrocytes (PCEs)
- Percentage of RETs or PCEs/normochromatic erythrocytes (NCEs)
- Bone marrow cell harvesting procedure (foetal calf serum, centrifugation at 300 g for 5 min)
- Slide preparation (air-drying, fixing in 90% methanol at -20 °C for 20 min, staining with acridine orange for 2 min)
- Microscopy (UV light, Olympus BX50 fluorescent microscope, 1000x magnification)
- Scoring (about 1000 RETs or PCEs per slide, coded and scored blindly)
- Positive control: Not specified
- Negative control: Not specified
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