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…
Disclosures
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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Cite This Article
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).