An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

Summary

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells' mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes – Hoechst 33342 and propidium iodide.

Abstract

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

Introduction

The first step to identifying effective cancer treatments is the selection of a robust, unbiased cytotoxicity assay that can be used to examine the effect of treatment. A common choice for low-throughput experiments is the exclusion of trypan blue dye from living cells. This method is favored because it allows a relatively unbiased method for quantifying cell survival. trypan blue passively diffuses into cells whose membranes are compromised, but it is effectively blocked from entering healthy cells¹. The quotient of the living cells and the total cells represents the percent viability, which indicates the efficacy of the treatment. The most si....

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 cytotoxicity of a single compound (e.g., to determine effective doses), prepare compounds at 2x final concentration.
   2. To measure 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. Collect c....

Results

The aforementioned protocol has been developed using OCI-AML2 cells, which were taken as a representative acute myeloid leukemia cell line. AML is characterized by abnormal proliferation of undifferentiated and non-functional hematopoietic cells in the bone marrow²⁶. Despite recent developments in AML targeted therapy, the standard of care has remained unchanged for several decades, and consists of induction therapy (typically comprised of three days of anthracycline, e.g., daunorubicin, idar.......

Discussion

Although the protocol for Hoechst/PI cytotoxicity assay is robust and requires comparatively little hands-on time, there are several experimental details that are very important to ensure accurate results. First, it is essential to make sure that the concentration of DMSO remains below 0.5% (v/v). It is generally agreed that exposure to even low doses of DMSO can substantially alter the morphology and attachment of cells and significantly delay cell cycle progression^39,

Materials

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
Alamar blue HS cell viability reagent (100mL)	Thermo Fisher	A50101
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
Pierce LDH assay kit	Thermo Fisher	50-103-5952
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
Thiazolyl blue tetrazolium bromide	ACROS Organics	AC158990010
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