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

Published: April 30, 2023

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 Prepare solutions of compounds of interest at desired concentrations in the appropriate media (serum-free or 1, 2.5, or 5% FBS RPMI-1640). To measure the cytotoxicity of a single compound (e.g., to determine effective doses), prepare compounds at 2x final concentration. To measure the cytotoxicity of compound combinations, prepare compounds at 4x final concentration. Prepare solvent-only controls by mixing the same amount of solvent with the appro…

Divulgations

The authors have nothing to disclose.

Materials

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

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Differential Nuclear Staining Assay: An Assay to Determine Mitocan Cytotoxicity by Propidium Iodide and Hoechst Double Staining. J. Vis. Exp. (Pending Publication), e21178, doi: (2023).

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