Cancer Side Population Discrimination Assay: A Method to Isolate Cancer Stem-like Cells Using Hoechst Dye Efflux Assay

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).

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	–