Fluorescently Labeled T-ALL Cells Isolation: A Method to Isolate T-ALL Cells from Adult Zebrafish

Abstract

Source: Pruitt, M. M., et al. Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish. J. Vis. Exp. (2017).

This protocol describes the technique of isolating fluorescently labeled T-cell acute lymphoblastic leukemia (T-ALL) tumor cells from an adult zebrafish and quantifying them using trypan blue dye.

Protocol

1. Collection of fluorescently labeled T-ALL cells from the primary tumor fish

1. Prepare at least 20 mL of 10% heat-inactivated fetal bovine serum in 0.9x phosphate buffered saline (FBS/PBS).
2. Add 1 mL of FBS/PBS to one vial of heparin sodium salt (300 units).
3. Select fish bearing tumors in more than 50% of the body, or those fish having difficulty eating or swimming. Sacrifice the fish by overdosing them with tricaine or by ice water immersion. Confirm death by lack of gill movement and heartbeat.
4. Use a razor blade to remove the head of the fish.
5. Wash the cavity of the fish with the heparin solution to remove excess red blood cells.
   1. Using a pipette, inject 100 µL of heparin solution (Add 1 mL of FBS/PBS to one vial of heparin sodium salt) into the body cavity of the fish. Remove all liquid that spills out (it will contain blood cells) and pipette it into a 1.5 mL microcentrifuge tube.
   2. Use a fresh pipette tip to wash the body again. Perform at least three washes, or until the liquid that spills out appears free of blood.
   3. Discard all liquid from the washes in these steps, as it will not be used in further analyses.
6. Collect the leukemia cells.
   1. Inject 100 µL of FBS/PBS into the body cavity of the fish.
   2. Apply gentle pressure to the outside of the body of the fish with the pipette tip to squeeze/push out the tumor cells.
      NOTE: If this step is performed using a dissecting microscope, the tumor cells spilling out of the body cavity will be evident.
   3. Collect the liquid and cells from each wash into a 1.5 mL microcentrifuge tube.
   4. Repeat steps 1.6.1-1.6.3 until most of the tumor cells are collected (usually 5-10 times). Collect all tumor cells into the same 1.5-mL microcentrifuge tube. Keep the cells at room temperature.
7. Mix the collected tumor cells from previous step by gently pipetting them to dissociate the clumps of cells. Filter the cell suspension through a 40-µm mesh filter. Wash the filter 1-2 times with 100 µL of FBS/PBS. Keep the cells at room temperature.
8. Mix 2 µL of cell suspension with 18 µL of trypan blue (0.4%, diluted 1:10 and filtered) in a new tube. Load 10 µL on a hemocytometer and count the number of live (non-blue) fluorescent cells to calculate the number of isolated tumor cells.
   NOTE: Non-blue GFP-negative cells are likely normal, non-malignant T-cells and should not be counted. At least 2 x 10⁶ cells are required to stain the cells with Hoechst 33342.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Syngeneic zebrafish (CG2)			See Mizgireuv et al., 2006
rag2:c-myc plasmid			Langenau Laboratory
rag2:GFP plasmid constructed in de Jong Lab			rag2 promoter from Langenau Laboratory
pCS2-transposase plasmid			Chien Laboratory
SteREO Discovery V8 Microscope	Carl Zeiss Microscopy	495015-0008-000
Tricaine methanesulfonate (MS-222)	Western Chemical, Inc	Fisher Scientific - NC0342409	100 g
Fetal bovine serum (FBS)	HyClone Laboratories, Inc.	Fisher Scientific - SH3007103	500 mL
Phosphate-Buffered Saline (PBS)	Gibco by Life Technologies	1001-023	pH 7.4 (1x) - 500 mL
Heparin sodium salt	Sigma-Aldrich	2106	15 - 300 unit vials
40 µm mesh filter	Falcon Corning Brand	352340	Case of 50
Trypan blue	Sigma Life Science	T8154	20 mL - Solution (0.4%)