For the preparation of the donor plate transfer 500, 000 wild type CG1 zebrafish cells into a 15 milliliter conical tube. Add five milliliters of fish media to dilute the cell stock to 100 cells per microliter. Dispense 50 microliters of the cell suspension into each well of the 96-well plate and place the plate at four degrees Celsius until sorting.
Prepare no stain and single color controls for the setup of the sorting experiment. Pass the cell suspension of the controls and the samples through a 35-micron filter cap into a fax tube on ice. Centrifuge the 96-well plate at 2, 500 G at four degrees Celsius.
Carefully remove 45 microliters of the supernatant from each well, leaving five microliters of liquid behind. Resuspend the cells with a 20-microliter pipette and using the Hamilton micro syringe, inject the cell suspensions into the desired number of CG1 zebrafish. After transplanting CG1 zebrafish with sorted cells for the limiting dilution analysis, the CT-FR high cells had a fourfold higher leukemic stem cell frequency compared to CT-FR low cells.
Representative images of zebrafish at 28 days post-transplant showed that 89%of CT-FR high zebrafish develop leukemia compared to 20%in the CT-FR low group. CT-FR high zebrafish had a significantly shorter latency to leukemia. CT-FR high zebrafish had a significantly lower overall survival compared to the CT-FR low zebra fish.
