The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis

Abstract

The MTT assay for cellular metabolic activity is almost ubiquitous to studies of cell toxicity; however, it is commonly applied and interpreted erroneously. We investigated the applicability and limitations of the MTT assay in representing treatment toxicity, cell viability, and metabolic activity. We evaluated the effect of potential confounding variables on the MTT assay measurements on a prostate cancer cell line (PC-3) including cell seeding number, MTT concentration, MTT incubation time, serum starvation, cell culture media composition, released intracellular contents (cell lysate and secretome), and extrusion of formazan to the extracellular space. We also assessed the confounding effect of polyethylene glycol (PEG)-coated gold nanoparticles (Au-NPs) as a tested treatment in PC-3 cells on the assay measurements. We additionally evaluated the applicability of microscopic image cytometry as a tool for measuring intracellular MTT reduction at the single-cell level. Our findings show that the assay measurements are a result of a complicated process dependant on many of the above-mentioned factors, and therefore, optimization of the assay and rational interpretation of the data is necessary to prevent misleading conclusions on variables such as cell viability, treatment toxicity, and/or cell metabolism. We conclude, with recommendations on how to apply the assay and a perspective on where the utility of the assay is a powerful tool, but likewise where it has limitations.

Keywords: MTT assay; PC-3 cells; cell metabolism; cytotoxicity; gold nanoparticles; how to; image cytometry; interpret; viability assay.

Figure 1

Factors affecting the final optical density (OD) measurements in the MTT assay. These include the concentration of MTT reagent and the proportion that actually enters the cell, cellular metabolic activity (which is highly dependent on a multitude of variables including treatments to the cells, biological effect of culture media, cell density, and impedance of cell metabolism due to toxic effects of MTT), cell number, timing of formazan crystals extrusion (which could impede further MTT uptake), chemical interference such as abiotic reduction of MTT by culture media, the tested treatment, or released cellular content, optical interference by all the background components, time of incubating cells with MTT reagent and/or tested treatment, and ultimately the optical measurement. Chemical structure of MTT and formazan are illustrated inside the cell: MTT consists of a tetrazole ring core containing four nitrogen atoms (1) surrounded by three aromatic rings including two phenyl moieties (2) and one thiazolyl ring (3). Reduction of MTT results in disruption of the core tetrazole ring and the formation of formazan. Red arrows and the “-“ sign indicate disruption of MTT reduction on the normal metabolic activity of the cells and the impeding effect of the formazan crystals (when presenting on the cell surface) on further uptake of MTT reagent by cells.

Figure 2

OD depends on cell seeding number/density, MTT concentration, and incubation time: OD changes with increasing cell seeding number/density. PC-3 cells were allowed to grow for 26 h before MTT addition. Absorbance was measured following 2 h (a), 3 h (b), and 4 h (c) of incubating cells with different concentrations of MTT. Data shown as mean OD of triplicate wells and error bars represent standard deviation (SD).

Figure 3

OD depends on cell seeding number/density, MTT concentration, and incubation time: OD changes with increasing MTT incubation time. (a) OD of cell-free culture media (Phenol red-containing RPMI + 10% FCS) at different time points after incubation with different concentrations of MTT. (b–d) OD of PC-3 cells with different seeding numbers of 5000 (b), 10,000 (c), and 20,000 (d) per well of 96-well plates. Cells were allowed to grow for 26 h before the addition of different concentrations of MTT and the OD was measured at different time points following MTT addition. Data shown as mean OD of triplicate wells and error bars represent SD (standard deviation).

Figure 4

OD depends on cell seeding number/density, MTT concentration, and incubation time: OD changes with increasing MTT incubation time. PC-3 cells were allowed to grow in 96-well plates for 26 h before incubation with MTT. OD was measured at different time points after addition of 10 µL of (a) PBS, or (b) 0.1 mg/mL, (c) 0.2 mg/mL, (d) 0.3 mg/mL, (e) 0.4 mg/mL, and (f) 0.5 mg/mL of MTT–PBS solutions to 100 µL of each well content. Data shown as mean OD of triplicate wells and error bars represent standard deviation (SD).

Figure 5

The optimum OD measurement time depends on how the OD levels change as a function of incubation time in all the tested conditions. A hypothetical graph showing OD levels as a function of MTT incubation time in two different conditions A and B. Each condition has resulted in a different saturation level and saturation time point and thus measurement time points (t1, t2, and t3 defined by dotted lines) that can affect the comparison of two conditions. Therefore, considering incubation time in comparative analysis gives us more accurate information on how each condition affects the behavior of the cell over time.

Figure 6

OD depends on cell seeding number/density, MTT concentration, and incubation time: OD changes with increasing MTT concentration. PC-3 cells were seeded at different numbers in 96-well plates and were allowed to grow for 26 h before adding 10 µL MTT–PBS solution of different concentrations (0–0.5 mg/mL) to 100 µL of each well content. OD was measured following 2 h (a), 3 h (b), and 4 h (c) of incubating cells with MTT. Data shown as mean OD of triplicate wells and error bars represent SD (standard deviation).

Figure 7

Intact, viable cells were necessary for MTT reduction. OD by lysate and supernatant of PC-3 cells compared to PC-3 cells (as positive control) and cell-free media (as negative control) following 3 hours of incubation with 0.4 mg/mL MTT. 20,000 cells were seeded per well of 96-well plates and were allowed to grow in phenol red-free FCS-containing RPMI for 24 h. The media was then replaced with fresh media of the same type with or without FCS. Two hours later, cells supernatant and lysates were isolated, and all samples had MTT added. Data shown as mean OD of triplicate wells and error bars represent SD (standard deviation). *** p-value < 0.0001.

Figure 8

Washing cells after incubation with MTT removes extracellular formazan crystals: The effect of washing the cells’ supernatant on the OD levels. 20,000 PC-3 cells were seeded per well of 96-well plates and were allowed to grow in phenol red-free RPMI with or without FCS for 26 h. The cells were then incubated with 0.4 mg/mL MTT for 3 h. Wells were then divided into two groups: (i) Non-washed wells: 85 µL of supernatant in each well was removed before adding DMSO so that 25 µL was left in each well. (ii) Washed wells: Supernatant was totally removed. Then wells were washed two times with PBS and 25 µL of fresh media was added before adding DMSO. Data shown as mean OD of triplicate wells and error bars represent standard deviation. * One-tail t-test p-value = 0.025.

Figure 9

Washing cells following MTT incubation removes intracellularly formed extracellular formazan. Light microscopy images (×4) showing the effect of washing the cells’ supernatant before solubilizing formazan on removing formazan crystals. 20,000 PC-3 cells were seeded per well of 96-well plates and were allowed to grow in phenol red-free RPMI with FCS for 26 h. The cells were then incubated with 0.4 mg/mL MTT for 3 h. The wells were then divided into 2 groups: (a) Non-washed wells, (b) Washed wells in which the supernatant was totally removed, and then cells were washed two times with PBS and fresh media (with the same volume as non-washed wells) was added to each well. Red arrows show the extracellular needle-shaped formazan crystals which are much fewer than the non-washed sample.

Figure 10

Optical and chemical interference by culture media components. (a) UV-Vis absorbance spectrum of RPMI with and without phenol red (PR) or 0.4 mg/mL MTT, (b) UV-Vis absorbance spectrum of RPMI without and with 10% fetal calf serum (FCS). Grey regions indicate the analysis window for OD measurements. Measurements in UV-Vis (a,b) were made against PBS for background, (c) OD of cell-free RPMI/PBS incubated with different concentrations of MTT for 2 to 4 h, (d) OD of cell-free RPMI with and without PR and/or FCS (10%) incubated with MTT-free PBS or 0.4 mg/mL MTT–PBS solution for 3 h before OD measurements in the MTT assay, (e) Effect of phenol red on MTT assay results on PC-3 cells. 20,000 PC-3 cells were seeded per well of 96-well microplate and allowed to grow for 26 h before 3 h of incubation with 0.4 mg/mL MTT. Corresponding cell-free media were used as negative control, (f) The OD of RPMI with and without FCS, incubated with MTT-free PBS or 0.4 mg/mL MTT –PBS solution for 3 h before OD measurements. Data shown as mean OD of triplicate wells and error bars represent standard deviation.

Figure 11

Serum starvation can affect MTT reduction. 20,000 PC-3 cells were seeded per well of 96-well plates and grown in phenol red-free RPMI with 10% FCS for 24 h. The media was then replaced with serum-free or serum-containing (10%) RPMI and cells were incubated for 2 h (a) or 26 h (b). Cells were then incubated with 0.4 mg/mL MTT for 3 h before OD measurements. Data shown as normalized mean OD of triplicate wells and error bars represent standard deviation. ** p-value = 0.005.

Figure 12

Extremely serum-starved cells show apoptotic morphologic features, yet still reduce MTT to formazan. Light microscopy images (×4) comparing cell morphology and formazan formation in serum-fed (a) and 26-h serum-starved cells (b) as per Figure 11. The purple color of cells is a result of formazan aggregation inside the cells. Red arrows show the extracellular needle-shaped formazan crystals which are much fewer in serum-starved cells.

Figure 13

Au-PEG NPs can interfere with MTT assay measurements. (a) UV-Vis absorption spectrum of RPMI with and without 0.2 nM Au-PEG NPs. Grey regions indicate the analysis window for OD measurements in the MTT assay. Measurements in UV-Vis were made against PBS for background, (b) OD of cell-free phenol red-free FCS-free RPMI containing different concentrations of Au-PEG NPs after 3 h of incubation with MTT-free PBS or 0.4 mg/mL of MTT–PBS solution, (c) OD of PC-3 cells treated with Au-PEG NPs compared to their lysate, supernatant, and cell-free culture media. 20,000 PC-3 cells were seeded per well of 96-well plates in phenol red-free RPMI with 10% FCS for 24 h. Media was then replaced with serum-free RPMI containing different concentrations of Au-PEG NPs and cells were incubated with NPs for 2 h. The supernatant of some cells was then removed and loaded in separate wells for MTT incubation. The remaining cells were lysed using the RIPA (Radioimmunoprecipitation assay) lysis buffer and covered with the same volume of NP-free media. The wells containing cells, cell lysate, cell supernatant, and cell-free culture media were then incubated with 0.4 mg/mL MTT for 3 h before OD measurements, (d) The fold change in OD levels for PC-3 cells in Figure 13c compared to their corresponding cell-free culture media with the same concentrations of Au-PEG NPs. The same data is presented as dark bars for cells incubated with the same concentrations of Au-PEG NPs in serum-containing media (RPMI + 10% FCS). Data shown as mean OD of triplicate wells and error bars represent SD (standard deviation). ** p-value < 0.005, *** p-value < 0.0001.

Figure 14

The heterogeneous location and morphology of intracellularly formed MTT-derived formazan. Light microscopy image (×20) showing formazan aggregates in PC-3 cells incubated with 0.4 mg/mL MTT for 3 h. Purple formazan aggregates are granular inside the cells (dashed arrows), but needle-shaped when located on the cell surface (red arrows), or suspended in the supernatant (stars).