Preparation of Test Subjects

I. Preparation of Test Subjects

The test subjects of the study were 32 white mice Mus musculus of ages 6-8 weeks old. The white mice were female and weighed 10-20 ± 1 g each. C.1 Maintenance and Feeding of Test Subjects The white mice were housed in a cage for the whole duration of the experiment. There were eight mice cages of dimensions 7.5‖ x 10‖ x 5‖ L x W x H; only one subgroup is to one cage. The cages were made of wood, aluminum wire, and screen. The cages also had some accessories which included a nest box, gravity-fed water bottle, and a food bowl. The cages were labeled as A, B, C, and D for the four subgroups of both the Normal Group and the Thrombocytopenic Group. The white mice were caged inside the Research Laboratory in Philippine Science High School – Western Visayas Campus for the whole duration of the study. All the subgroups were fed with mice pellets in ad libitum every day. Feeding was served on a free-choice basis. Plenty of fresh, clean water in feeding bottles was also available to the white mice at all times. C.2 Assignment of Test Subjects The white mice were assigned randomly to each subgroup using random assignment. They were put in a common container, picked one by one, and put one by one into each subgroup‘s cage in this order: Normal A, Normal B, Normal C, Normal D, Thrombocytopenic A, Thrombocytopenic B, Thrombocytopenic C, and Thrombocytopenic D. C.3 Preparation of the Thrombocytopenic Group The method used by Crow and others 2003 was adapted in this study to induce thrombocytopenia to the white mice. Two µg of the 0.5 mgml rat anti-white mice antiplatelet antibody MWReg30 already in 200 µL Phosphate-buffered saline PBS of pH 7.2 from Genomax Technologies, Singapore was injected to the white mice intraperitoneally. The white mice were restrained properly refer to Appendix A and were held at a slightly downward angle. The surface of the area to be injected was disinfected first by rubbing cotton in 75 alcohol on the area. Then, the 26G ½‖ needle of the 1 mL syringe was injected on the left side of the white mice, halfway between the midline and the top of the hind leg, at approximately 0.5 cm deep into the abdominal cavity and at an angle of 15 -20. The solution was then injected slowly into the white mice at a constant rate. After that, the needle was removed and the white mice were put down gently. The white mice will have to become thrombocytopenic after 24 hours according to Crow and others 2001.

J. Collection of Blood Samples