Side Population Assay: A Method to Isolate Side Population of Tumor Cells from Zebrafish
Published: April 30, 2023
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 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. 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 …
Offenlegungen
The authors have nothing to disclose.
Materials
| 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 |
Diesen Artikel zitieren
Side Population Assay: A Method to Isolate Side Population of Tumor Cells from Zebrafish. J. Vis. Exp. (Pending Publication), e20281, doi: (2023).