Effect of quercetin on the morphology of chicken embryo blood cells

Description

Chicken-embryo blood cells were assessed for morphological differences upon incubation with solvent control (DMSO), quercetin, methyl methanesulfonate (MMS), cadmium chloride (CdCl2) and phosphate buffered saline (PBS). After incubation, blood was extracted, transferred to glass slides, and blood smears were performed. Dry blood smears were fixed and sequentially stained with eosin Y and a mixture of methylene blue/azure B. After washing, blood smears were mounted, and slides were imaged by bright-field microscopy (40 x magnification).

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Fertilized chicken eggs (Ross 308, minimum weight 50 g) were inspected by candling to confirm integrity. Eggs containing viable embryos were washed with sterile distilled water and incubated in a humidified atmosphere (70 % humidity) at 38 °C. Eggs were maintained at 45° angle relative to the horizontal axis (pointed end facing down), and routinely rotated 90° around the central vertical axis, three times per day during the first 7 days (days 1 – 7), to prevent blood vessel and embryo attachment to any of the sides of the egg. At the beginning of day 7, egg position was changed from 45° to 90° angle (fully vertical, pointed end facing down), to allow the uniform position of the air cell at the blunt egg top. Quercetin was dissolved in DMSO and further dissolved to final concentrations of 667, 66.7 and 6.67 µg/ml, in PBS (1% DMSO final concentration in all conditions). MMS (3.67 mg/ml) and CdCl2 (133 µg/ml) were dissolved in PBS containing 1% DMSO. All solutions were prepared freshly before addition. At the beginning of day 8, after candling to discard non-viable embryos and mark the air cell area, a small opening was created on the egg top, to allow pipetting of each solution. Each egg was inoculated on the inner membrane with 150 µl of each solution, scotch tape was applied, and the eggs were immediately placed in the incubator. At the beginning of day 11, curved dissecting scissors were used to create an opening on the egg top, within the limits of the air cell. The inner membrane was wetted with PBS solution and carefully opened with forceps. A small plastic strip was then gently inserted under a larger blood vessel, to rise the vessel. The exposed vessel was gently washed with PBS, blot-dried with paper tissue, and carefully incised. Blood (200 µl) was immediately harvested with micropipette and placed on a glass slide. A blood smear was performed and each slide was given an alphanumerical code for subsequent blind scoring of cells. After drying at room temperature, blood smears were fixed, stained, and washed according to Hemacolor kit (Sigma-Aldrich, St. Louis, MO-USA) protocol. Blood smear samples were mounted with Entellan new (Sigma-Aldrich, St Louis, MO-USA), and stored until visualization by microscopy. Microscopic slides were visualized by bright-field microscopy at 40 × magnification (10 × ocular lens, 4 × objective lens).

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