Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Summary

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

Abstract

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

Protocol

MCF-7 seeding in 96-well plate:

MCF-7 cells were cultured in 25 t-flask in medium containing Dulbecco’s Modified Eagle’s Medium (DMEM), 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO₂, 95% air and complete humidity. Once reached ~90% confluency, they were detached using 0.05% trypsin/EDTA and counted by means of trypan blue and hemocytometer. These cells were then resuspended at a concentration of 4×10⁴ cells/cm² and added onto 96-well plate (i.e., 250 μl/well) by an 8-channel pipette. For background absorption, some wells were remained cell-free, i.e. as blank control.

Treating cells with different nanopolyplexes:

At 40-50% confluency (48 hours post seeding), the cultivated cells were treated with nanostructured starburst polyamidoamine dendrimers (i.e., Superfect® and Polyfect®) and a novel test polymer following the transfection instruction provided by supplier. Cells were also treated with EGFR and scrambled antisense alone, and with the three different nanopolyplexes of these two oligonucleotides and with polymers (n=4). Four wells were remained untreated as control. After 4 hours the treatment media were removed and replenished with fresh media.

MTT assay for evaluating cell viability:

MTT assay was performed 24 hours after transfection. For this purpose, MTT solution was prepared at 1mg/ml in PBS and was filtered through a 0.2 µm filter. Then, 50 µl of MTT plus 200 µl of DMEM without phenol red were added into each well, except the cell-free blank wells. Cells were incubated for 4 hours at 37°C with 5% CO₂, 95% air and complete humidity. After 4 hours, the MTT solution was removed and replaced with 200 µl of DMSO and 25µl Sorenson’s glycine buffer (glycine 0.1M, NaCl 0.1M, pH:10.5 with 0.1 NaOH). The plate was further incubated for 5 min at room temperature, and the optical density (OD) of the wells was determined using a plate reader at a test wavelength of 570 nm and a reference wavelength of 630 nm.

Discussion

The MTT assay is deemed to be a versatile method and accordingly the viability of the cells could be evaluated upon various treatments. The production of resultant formazan appears to be proportional to the level of energy metabolism in the cells. Therefore, it is possible to measure the metabolically activated cells even in the absence of cell proliferation. The amount of formazan produced is proportional to the amount of MTT in the incubation medium. While, the concentration of MTT which is required to achieve maximum ...

Materials

Name	Company	Catalog Number	Comments
25 t-flask	Orange Scientific	5510100
PBS	Sigma-Aldrich	P3813
Dulbecco’s Modified Eagle’s Medium - high glucose	Sigma-Aldrich	D5671
MTT	Sigma-Aldrich	M2128
FBS	Sigma-Aldrich	F2442
Penicillin 200,000 u/ml	CinnaGen	CR7913
Streptomycin 200 mg/ml	CinnaGen	CR7912
trypsin/EDTA	Sigma-Aldrich	T3924
trypan blue	Sigma-Aldrich	T8154
Superfect	Qiagen	301105
Polyfect	Qiagen	301305
EGFR antisense	Eurofins MWG Operon
Scrambled antisense	Eurofins MWG Operon
DMEM without phenol red	GIBCO, by Life Technologies	11880
Glycine	Sigma-Aldrich	G8898
hemocytometer	HBG
8-channel pipette	TreffLab Transferpette	96.9918.10.1
Disposable syringe filter	Iwaki, Asahi Techno Glass Corp.	2052-025
Plate reader equipped with 570 nm &630 nm filters	BioTek	ELX808
96-well plate with lid (flat bottom)	Iwaki, Asahi Techno Glass Corp.	3860-096
Laminar flow hood	Esco Products
CO2 Incubator	ASTEC