Cancer Side Population Discrimination Assay: A Method to Isolate Cancer Stem-like Cells Using Hoechst Dye Efflux Assay
Published: April 30, 2023
Abstract
Source: Gilormini, M. et al. Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics. J. Vis. Exp. (2016)
In this video, we describe the isolation of a subpopulation of cancer stem-like cells from head and neck squamous carcinoma cell lines using fluorescence-activated cell sorter or FACS. The isolated side population of cells can be used for further downstream analysis.
Protocol
1. Selection of a Side Population (SP) by the Hoechst Dye Efflux Assay Staining 50 million cells with Hoechst 33342 dye Prepare two 15 mL sterile tubes with a conical bottom: one tube labeled "Hoechst", and the other labeled "Hoechst and Verapamil". Prepare 10 mL of 5 mM Verapamil hydrochloride solution in sterile water. Prepare the culture medium (CM) for stem cells. To prepare CM for CSC (CM-CSC), combine the following: Dulbecco's modified Eagle…
Representative Results
Figure 1: Isolation of a side population excluding the Hoechst dye. (A) FSC-A versus SSC-A dot plot. (B) SSC-W versus SSC-H dot plot. (C, D) Hoechst Red-A versus Hoechst Blue-A dot plot using the "Hoechst" tube (C) or with the "Hoechst and Verapamil" tube (D).
開示
The authors have nothing to disclose.
Materials
| Fetal Calf Serum Gold | GE Healthcare | A15-151 | |
| Hydrocortisone water soluble | Sigma-Aldrich | H0396-100MG | |
| Penicillin/Streptomycin 100x | Dominique Dutscher | L0022-100 | |
| DMEM | Gibco | 61965-026 | |
| F12 Nut Mix (1x) + GlutaMAX-I | Gibco | 31765-027 | |
| EGF | Promega | G5021 | The solution must be prepared just before use because it is very unstable |
| Heparin | StemcellTM Technologies | 7980 | |
| B-27 Supplement (50x), minus vitamin A |
Gibco | 12587-010 | |
| Penicillin/Streptomycin 100x | Dominique Dutscher | L0022-100 | |
| DMEM | Gibco | 61965-026 | |
| B-27 Supplement (50x), minus vitamin A |
Gibco | 12587-010 | |
| Hoechst 33342 | Sigma-Aldrich | 14533 | Corrosive, acute toxicity (oral, dermal, inhalation) category 4 |
| Verapamil hydrochloride | Sigma-Aldrich | V-4629 | Acute toxicity (oral, dermal, inhalation) category 3 |
| Propidium Iodide | Sigma-Aldrich | P4170 | Acute toxicity (oral, dermal, inhalation) category 4 |
| SQ20B cell line | Gift from the John Little’s Laboratory |
– | |
| FaDu cell line | ATCC | HTB-43 | |
| BD FACSDiva software v8.0.1 | BD Biosciences | – |
