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 Prepare at least 20 mL of 10% heat-inactivated fetal bovine serum in 0.9x phosphate buffered saline (FBS/PBS). Add 1 mL of FBS/PBS to one vial of heparin sodium salt (300 units). 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 m…

Materials

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%)