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

Overview

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 verapamil is added to the sample.
3. Set a water bath to 28 °C.
4. Prepare samples and controls in the dark by adding cells and solutions to fluorescence-activated cell sorter (FACS) tubes.
   1. Prepare the samples by adding 10⁶ cells, 15 µL of 1 mg/mL Hoechst 33342, and 2.5 µL of DMSO to a FACS tube. Adjust the volume to 1 mL with FBS/PBS.
   2. Prepare the controls by adding 10⁶ cells, 15 µL of 1 mg/mL Hoechst 33342, and 2.5 µL of 100 mM verapamil to a FACS tube. Adjust the volume to 1 mL with FBS/PBS.
      NOTE: A minimum of 10⁶ cells/sample is recommended, but staining more cells is preferred, especially if the side population cells will be sorted for further analysis. Do not exceed 10⁶ cells/mL, and maintain the Hoechst 33342 concentration at 15 µg/mL. If 10 x 10⁶ cells are stained, the total reaction volume is 10 mL.
5. Incubate in a 28 °C water bath in the dark for 120 min.
6. After the incubation, place the cells immediately on ice and keep them in the dark.
7. Pellet the cells at 4 °C for 6 min at 300 x g. Remove the supernatant. Wash with 1 mL of FBS/PBS.
8. Pellet the cells at 4 °C for 6 min at 300 x g. Remove the supernatant. Resuspend the cells in 1 mL of FBS/PBS.
9. Keep at 4 °C until cell sorting.
10. 15 min prior to cell sorting, add 2 µL of propidium iodide (PI) stock (1 mg/mL) to each 1 mL of FBS/PBS (final concentration: 2 µg/mL). Mix well.

2. Use FACS to Find the Side Population within the Fluorescently Labeled T-ALL Cell Population

1. Analyze the stained zebrafish T-ALL cell suspensions on a cell sorter equipped with a UV laser for Hoechst 33342 dye excitation and a 488-nm laser for PI excitation and for the detection of the GFP+ T-ALL cells.

Materials

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