Hematopoietic and Vascular System Toxicity - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 77

seen from cytospin analysis, while allowing study of much higher blood cell
numbers (Traver et al., 2003b).

Flow Cytometry Analysis

1. Isolate kidney marrow as described above.
2. Add 1 L propidium iodide (PI), which is incorporated by dead cells, to

filtered marrow cells.
3. Perform analysis on a flow cytometer. Analyze PI levels and light scatter
characteristics.
4. Remove PI positive cells (dead cells) from analysis.
5. Create gates based on forward and side scatter characteristics.

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7.3.3 Functional HSC and Blood Progenitor Assays

Looking at gross morphology or blood cell number in the adult zebrafish does not
prove that HSCs are unaffected by chemical treatment. Unlike many mammalian
systems, it is not possible to sort out HSCs from the zebrafish kidney marrow using
stem cell-specific antibodies. However, the presence of HSCs can still be tested by
functional transplantation assays. As shown in mammalian models (Uchida
et al., 1994), zebrafish HSCs are capable of reconstituting the entire hematopoietic
system. In particular, they can rescue the blood system in a fish treated with a lethal
dose of irradiation (Traver et al., 2004) and contribute to reconstitution of blood
cells in a sublethally irradiated fish (White et al., 2008). If a chemical-treated adult
fish has maintained functional HSCs, then it will also be able to rescue the
hematopoietic system. This assay may primarily be useful for testing a select
number of compounds.

Transplant Assay

1. Chemically treat adult glo-fish (available from S-D Tropical and Segrest
Farms) (or an alternate transgenic fish with fluorophore-labeled blood cells)
for the desired length of time. Alternatively, cells can be treated ex vivo at step
3 for up to 4 h.
2. Irradiate wild-type untreated recipient fish at 30 Gy evenly split between one
and two days pre-transplant.
3. Remove kidneymarrow cells as described above from chemically treated fish.
4. Count cells using a hemacytometer or an alternate approach.
5. Via intracardial or retro-orbital injection, inject 100,000 kidney marrow cells
into each irradiated recipient fish (2 days post irradiation).
6. Four weeks post transplant, isolate kidney marrow from recipient fish.
7. Analyze recipient fish for fluorophore (i.e., GFP) positive cells in the marrow.
In particular, analyze the light scatter characteristics of GFP positive cells. If
HSCs in the chemical treated fish or cells were functional, then GFP positive
cells will be present in the population (White et al., 2008).