Differential Nuclear Staining Assay: An Assay to Determine Mitocan Cytotoxicity by Propidium Iodide and Hoechst Double Staining

Abstract

Source: Pei, J. et al., An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity. J. Vis. Exp. (2020)

This video demonstrates a nuclear staining method to detect the cytotoxic effect of mitocan on drug-sensitive cancer cells and healthy variants. The double staining with DNA-binding dyes such as propidium iodide and Hoechst dye provides a clear distinction between the dead and live cells, thus helping to assess the cytotoxicity of the drug.

Protocol

1. Cytotoxicity Assay: Setup

1. Prepare solutions of compounds of interest at desired concentrations in the appropriate media (serum-free or 1, 2.5, or 5% FBS RPMI-1640).
   1. To measure the cytotoxicity of a single compound (e.g., to determine effective doses), prepare compounds at 2x final concentration.
   2. To measure the cytotoxicity of compound combinations, prepare compounds at 4x final concentration.
   3. Prepare solvent-only controls by mixing the same amount of solvent with the appropriate medium. For example, if testing compounds dissolved in DMSO and methanol, make solvent-only control for each solvent.
2. C....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Deoxy-D-glucose/2-DG	Chem-Impex	50-519-067
3-bromo-pyruvate	Alfa Aesar	1113-59-3
96-Well plates	Greiner Bio-One	655090	Black or clear flat-bottomed 96-well plates
Countess II automated cell counter	Thermo Fisher
Cytation 5 Cell Imaging Multi-Mode Reader	BioTek
Hoechst 33342	Thermo Fisher	62249	20 mM solution; final concentration 1:1,000
HyClone fetal bovine serum	GE Healthcare	#25-514
m-chlorophenylhydrazone/CCCP	Sigma Aldrich	C2759
PBS tablets	Thermo Fisher	BP2944100	1 tablet + 200 mL of sterile water = 1x PBS solution
Penicillin-Streptomycin-Glutamine (100X)	Gibco	10378016
Propidium Iodide	Thermo Fisher	50-596-072	Dry powder; stock 1 mg/mL in PBS; final concentration 5 µg/mL (leukemia cells), 1 µg/mL (normal PBMCs)
Rotenone	Ark Pharm	AK115691
RPMI-1640 Medium With L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture	Sigma Aldrich	R8758-500ML
Trypan blue stain (0.4%)	Gibco	15250-061
Cell lines
K562	ATCC	CCL-243	CML cell line
MOLM-13	ATCC		AML cell line
MOLT-4	ATCC	CRL-1582	ALL cell line
OCI-AML2	ATCC		AML cell line