Side Population Assay: A Method to Isolate Side Population of Tumor Cells from 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).

The following protocol describes a method for isolating the side population of T-cell Acute Lymphoblastic Leukemia (T-ALL) tumor cells, consisting of stem cells, from an adult Zebrafish. The side population lacks prominent cell markers and hence we use Hoechst 33342 dye efflux assay to isolate them.

Protocol

1. Staining the Fluorescently Labeled T-ALL Cells with Hoechst 33342 to Identify the Side Population

1. Dilute Hoechst 33342 1:10 with nuclease-free H₂O to make the 1 mg/mL working concentration. Store the dye at 4 °C and in the dark.
2. Prepare a 100-mM solution of verapamil by dissolving 49.1 mg in 1 mL of dimethyl sulfoxide (DMSO).
   NOTE: Verapamil is an inhibitor of ABC transporters and is an important control for side population experiments. Adding verapamil to samples will inhibit the ability of the ABC transporters to efflux the Hoechst 33342 dye. Therefore, a true side population will disappear when verapa....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Hoechst 33342	Life Technologies	H3570	10 mL
Verapamil	Sigma Life Science	V4629	1 g
Dimethyl sulfoxide (DMSO)	Sigma Life Science	D8418	100 mL
Propidium Iodide	Life Technologies	P3566	10 mL
FACSAria Fusion cell sorter	BD Biosciences