Zebrafish Xenograft Assay: A Method to Assess the Anti-cancer Efficacy of Therapeutics on Tumorigenicity in Zebrafish In Vivo
筆記録
– First, place the eggs into a Petri plate, and add Danieau's solution to maintain freshly collected eggs. Add phenyl thiourea to inhibit pigmentation and incubate for a few days. After incubation, dechorionate the embryos using sharp forceps and let them grow further.
Next, seed the desired number of cancer cells in a culture flask and incubate overnight. Now add anticancer compounds and further incubate. After incubation, add fluorescent cell tracker dye, which will bind to the cell membrane and stain the cells.
Next, add trypsin for cell dissociation and dilute the cells in the phenol red PBS solution before the procedure to assess pH. Mount anaesthetized zebrafish embryos on an agar plate. Now load the microcapillary with cancer cell suspension and inject it into the yolk sac of embryos.
Transfer embryos to culture plates containing Danieau's solution and incubate further. Next, transfer a few embryos on a glass slide and add methylcellulose to immobilize them. Place the slide under a fluorescence microscope and quantify the tumor size using the software.
Anticancer compounds usually inhibit the tumor forming capacity of cancer cells. In the following protocol, we will study the effect of hydroxycoumarin, along with BH3 mimetic, on tumor formation in zebrafish in vivo.
– After seeding A549 cells and incubating them with compounds according to the text protocol, 24 hours before the end of treatment, add four micromolar of the fluorescent cell tracker dye CM-Dil to stain the cells and incubate them for two hours.
Following the incubation, use 0.05% trypsin EDTA to trypsinize the cells. Then dilute 10 to the 6th cells in 50 microliters of phenol red PBS solution prior to injection. To carry out microinjection of cancer cells, pull 1.0 millimeter glass capillary using the following program settings.
Use a syringe to cut the capillary to produce a sharp edge. Then load the microcapillary with 20 microliters of the cell phenol red solution. After anesthetizing zebrafish according to the text protocol, inject 100 to 200 cells into the yolk sac by injecting 6 to 10 nanoliters via three to five injections.
– Microinjections should be done very carefully and precisely to ensure that the correct volume of cells is injected, and injection injury is minimal, and the carbon dioxide pressure should be optimal to avoid fish lethality.
– After injection, allow the fish to recover for 10 to 30 minutes in 5 milliliters of fresh Danieau's solution containing PTU. Transfer the fish into 24 well plates with one milliliter of Danieau's PTU solution and incubate them at 28.5 degrees Celsius for 72 hours. Following the incubation, after anesthetizing the fish according to the text protocol, immobilize them on a glass slide with a drop of 3% methyl cellulose.
