Optimization and In Vivo Propagation of CellTrace Far Red (CT-FR) Stained Zebrafish T-ALL Cells

To begin, collect one time 10 to the power of six fluorescently labeled zebrafish, T-ALL cells in 1.5 milliliter microcentrifuge tubes. Centrifuge the cells at 2, 500 G for five minutes at four degrees Celsius and resuspend the pellet in one milliliter of 0.9 XPBS. Place 250 microliters of cell suspension into a microcentrifuge tube.

Then, add 250 microliters of 0.9 XPBS to bring the volume up to 500 microliters. Add the required volume of CT-FR Dye stock solution and incubate for 20 minutes at 37 degrees Celsius, protecting from light. Centrifuge the cells at 2, 500 G for five minutes at room temperature.

Add 500 microliters of fish media to remove the excess dye and centrifuge again before resuspending the pellet in 25 microliters of fish media. Stain the cells with trypan blue and examine them under the microscope for viability. Using the optimized CT-FR concentration, stain the zebrafish cells and leave some tumor cells unstained.

Using a Hamilton microsyringe, inject five microliters of stained and unstained cell suspension into the intraperitoneal cavity of anesthetized zebrafish.