Hematopoietic and Vascular System Toxicity - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 76

of blood cells, peripheral blood or whole kidney marrow can be isolated and
visualized by cytospins.

Isolating Peripheral Blood from the Adult Zebrafish (Chan et al., 1997)

1. Anesthetize adult zebrafish in 4% 4 g/L tricaine.
2. Puncture the heart with a glass microhematocrit tube to collect blood cells.
3. Wash the cells in 0.9 PBS before characterization.

Isolating Kidney Marrow from the Adult Zebrafish

1. Euthanize adult zebrafish in 10-20% 4 g/L tricaine.
2. Dissect fish and remove kidney (LeBlanc et al., 2007).
3. Place kidney in 300-500

m L of 0.9 PBS and 5% fetal bovine serum solution.

4. Triterate using 1000 m L pipette tip until kidneymarrow has been disaggregated.

5. Pass through a 40 m m filter.

Cytospins

1. Dilute cells 1:10 in 0.4% filtered trypan blue. Trypan blue is incorporated by
dead cells, so they appear blue under the microscope.
2. Count live cells using a hemacytometer or other method.
3. Dilute (or concentrate) cells so that there are 100,000-200,000 cells in 600 m L.

4. In a cytospin cytocentrifuge, spin down 200 m L for 3min at 300 rpmonto glass

slides.
5. Wait overnight for slides to dry.
6. Stain with May-Grunwald and Giemsa as described above.

Although cytospin analysis provides morphological data, flow cytometry analysis is
an alternative approach to identifying the percentage of kidneymarrowcells of each
blood cell type. Using flow cytometry, cells can be analyzed by light scatter
characteristics, specifically forward scatter (FSC) and side scatter (SSC). FSC is
directly proportional to cell size, whereas SSC is directly proportional to cellular
granularity (Traver et al., 2003b). When whole kidney marrow is analyzed using
these characteristics, four populations can be identified (Fig. 7.2b). A population
with low FSC contains mature erythrocytes. A population with both high FSC and
SSC contains myeloid cells andmonocytes/macrophages. Lymphoid cells fall into a
population with low side scatter and intermediate forward scatter characteristics.
Myeloid, lymphoid, and erythroid precursors fall into a population in between the
myeloid and lymphoid populations. More recent analysis of CD41 expressing cells
demonstrates that HSCs, as well as thrombocytes, are present in the lymphoid gate
(Lin et al., 2005). An advantage of the flow cytometry approach is that cells can also
be sorted into the four populations using flow-activated cell sorting (FACS).
Overall, flow cytometry of zebrafish whole kidney marrow correlates with data