The mouse fibroblast cell line BALB/3T3 was obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from Life Technologies Corporation (Grand Island, NY, USA). Sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO), hydrochloric acid (HCl), Fetal Bovine Serum (FBS) and 0.25% trypsin-EDTA solution were obtained from Thermo Fisher Scientific (Cranbury, NJ, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit was purchased from Millipore Sigma (Rockville, MD, USA).

BALB/3T3 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 0.01 mg/mL streptomycin sulphate. Prior to experimentation, BALB/3T3 cells were subcultured to account for their adherent growth properties. The cells were rinsed with about 10 mL of phosphate-buffered saline (PBS, pH 7.4) after the unused medium was removed. The cells were then allowed to separate from the monolayer for a few minutes at room temperature after about 3 mL of 0.25% trypsin-EDTA solution in Hanks’ balanced salt solution (without calcium or magnesium) were added. The detached cells were then resuspended in complete medium, and cell viability was determined using the dye exclusion method [16]. The cells were maintained at greater than 90 percent viability throughout the study.

The MTT assay was employed to evaluate the metabolic activity of BALB/3T3 cells and to assess the effects of different solvents on formazan dissolution. The basis of this colorimetric analysis is the reduction of the yellow tetrazolium salt of MTT in metabolically active cells by the mitochondrial enzyme succinate dehydrogenase to a water insoluble formazan crystal. The amount of formazan produced is directly proportional to the number of viable cells and can be quantified spectrophotometrically at 570 nm.

To evaluate the influence of cell density and incubation time on formazan solubilization, BALB/3T3 cells were seeded in 100 μL volumes at densities of 2500, 5000, and 10,000 cells per well. The cells were cultured for overnight at 37˚C in a humidified incubator containing 5% CO₂. Following incubation, 10 μL of MTT stock solution (5 mg/mL) was added to each well of two separate 96 well plates, and the plates were incubated for an additional 4 h under the same conditions to allow MTT reduction and formazan crystal formation.

After incubation, the culture medium was removed from the wells of the first plate without disturbing the adherent cells and the formazan crystals, whereas the medium in the wells of the second plate was left undisturbed. Subsequently, 200 μL of various solubilization solvents such as DMSO, DMSO containing 0.005 N HCl, 10% SDS in water with 0.005 N HCl, 5% SDS with 0.005 N HCl in DMSO, or 10% SDS with 0.005 N HCl in DMSO was added separately to each well. The plates were then incubated under the same conditions at 37˚C for 1 h to ensure complete dissolution of the formazan crystals and the formation of homogeneous solutions. Absorbance of the resulting-colored solutions was measured at 570 nm. Background absorbance for medium-containing wells was corrected using blank wells filled with 100 μL of complete medium, while background absorbance for medium-free wells was adjusted using 100 μL of each solubilization solvent alone.

A suspension containing 5 × 10³ BALB/3T3 cells in 100 μL was dispensed into each well of two separate 96-well plates. In parallel, two cell-free control plates were prepared to evaluate potential non-enzymatic reduction of MTT by culture medium components, FBS, and the solubilizing solvents [6]. In the first control plate, each well was added with 100 μL of a different solubilization solvent: DMSO, DMSO containing 0.005 N HCl, 10% SDS in water with 0.005 N HCl, 5% SDS with 0.005 N HCl in DMSO, or 10% SDS with 0.005 N HCl in DMSO. In the second control plate, each well added 100 μL of complete medium, but without cells.

The test and control plates were incubated overnight at 37˚C in a humidified atmosphere containing 5% CO₂. Following incubation, 10 μL of MTT stock solution (5 mg/mL) was added to each well, and the plates were further incubated for 4 h under the same conditions. At the end of this period, the culture medium in the wells of the first test plate was carefully aspirated to ensure complete removal without disturbing either the adherent cells or the formazan crystals formed on the plastic surface. However, the medium in the wells of the second test plate had remained unchanged. For the control plates, both the medium and the solvents were retained without removal. Subsequently, 200 μL of different solubilization solvents such as DMSO, DMSO containing 0.005 N HCl, 10% SDS in water with 0.005 N HCl, 5% SDS with 0.005 N HCl in DMSO, and 10% SDS with 0.005 N HCl in DMSO were added individually to each test and control plates wells. The plates were then incubated at 37˚C for periods ranging from 5 min to 24 h to allow dissolution of the formazan crystals and formation of a homogeneous solution in the respective solvents. The absorbance of the colored solutions resulting from the different solubilization solvents was measured at 570 nm at each test time point. The background absorbance in medium-containing wells was adjusted using blank wells that contained 100 μL of complete medium. Similarly, background absorbance in medium-free wells was subtracted by using 100 μL of each solubilization solvent independently. These experiments used data from two or three replicate wells to calculate each point, and three independent assays were used to evaluate each test condition.

To examine the impact of HCl concentration in formazan solubilization solvents, BALB/3T3 cells were seeded into two separate 96-well plates at a density of 5000 cells per well in 100 μL of suspension. The plates were incubated for overnight at 37˚C in a humidified atmosphere containing 5% CO₂. After incubation, 10 μL of a 5 mg/mL MTT stock solution was added to each well, and the plates were incubated for an additional 4 h under the same conditions. Following incubation, the culture medium was carefully removed from the first plate’s wells without disturbing the cells or the formazan crystals that had developed, while medium had remained in the second plate’s wells. Then, 200 μL of the respective solubilization solvents were individually added to each well: DMSO, DMSO containing 0.01 N HCl, 10% SDS in water with 0.01 N HCl, 5% SDS in DMSO with 0.01 N HCl, and 10% SDS in DMSO with 0.01 N HCl. Afterward, the plates were incubated at 37˚C for varying time intervals ranging from 10 min to 24 h to ensure complete dissolution of the formazan crystals and the formation of homogeneous solutions under each solvent condition. The absorbance of the colored solutions resulting from the different solubilization solvents was measured at 570 nm at each test time point. Blank wells containing 100 μL of complete medium were used to adjust the background absorbance in medium-containing wells. Similarly, 100 μL of each solubilization solvent was used separately to subtract background absorbance in medium-free wells. Each point in these experiments was calculated using data from two or three replicate wells, and each test condition was assessed using two separate assays.

The assay is dependent on the ability of viable cells to metabolize a water-soluble tetrazolium salt into a water-insoluble formazan product. To examine the effect of cell density and incubation conditions on formazan solubilization, BALB/3T3 cells were seeded in 96-well plates at densities of 2500, 5000, and 10,000 cells per well in 100 μL volumes. Following incubation with MTT, two approaches were used: in one plate, the medium was completely aspirated without disturbing the cells or formazan crystals (Figure 1(A)), while in the other plate the medium was retained (Figure 1(B)). Subsequently, 200 μL of various formazan-solubilizing solvents such as DMSO, DMSO with 0.005 N HCl, 5% SDS and 0.005 N HCl in DMSO, 10% SDS and 0.005 N HCl in DMSO, and 10% SDS and 0.005 N HCl in water were added to each well, and the plates were incubated for 1 h at 37˚C. In theory, the intensity of the formazan color directly reflects the number of viable cells. The resulting OD values (Figure 1(A) and Figure 1(B)) demonstrated that OD increased with cell seeding density, reflecting the greater amount of formazan produced by larger cell populations. This result is consistent with earlier studies demonstrating that increasing cell counts lead to proportionally stronger MTT signals [17], while OD values begin to decline once cell numbers surpass a critical threshold [17]. When formazan solubilizing solvents are added directly to the culture medium, an increase in OD at 570 nm is typically observed (Figure 1(B)). In the present experiment, however, no saturation in OD was detected with rising cell density. This result is unsurprising, as the maximum seeding density employed (10,000 cells per well) corresponded to only ~70% - 80% confluence at the time of measurement (following overnight incubation), as confirmed by light microscopy observations.

To understand how assay parameters affect the relationship between OD and cell number, these results show that thorough control experiments are necessary prior to using the MTT assay to assess cell viability. Importantly, because formazan production depends on more than just the number of viable cells, OD values may still be biased. MTT uptake [18] and metabolic activity [19] can be changed by experimental conditions or treatments, which can impact the result. Since such, MTT reduction, formazan formation, and absorbance are all influenced by a variety of factors rather than just one, which is ultimately reflected in the measured OD. Results are means from three independent experiments. In subsequent assays, a seeding density of 5,000 cells per well was selected based on this prior cell density evaluations.

The present investigation tested whether the combination of DMSO, SDS, and HCl provides a synergistic impact on the dissolving of water-insoluble formazan, considering that DMSO and SDS lysis solutions are acknowledged as among the most effective solvents for formazan [9][10]. The choice of solubilizing solvents was guided by preliminary test results as well as evidence from the literature [20]. The suitability of SDS and the organic solvent DMSO for use in the MTT assay for BALB/3T3 cells was tested using a variety of formazan solubilization solvents, such as DMSO, DMSO with 0.005 N HCl, 5% SDS and 0.005 N HCl in DMSO, 10% SDS and 0.005 N HCl in DMSO, and 10% SDS and 0.005 N HCl in water. Since phenol red is frequently used as a pH indicator in cell culture medium. It might have an impact on formazan’s absorption spectrum in MTT colorimetric tests [15]. To mitigate this effect, 0.005 N HCl was added to the solubilizing solvents. At this acidic pH, all the phenol red is protonated and evenly yellow, reducing its spectral interference. This kind of the dye has little to no impact on absorbance measurements at an appropriate wavelength. The aim of this investigation was to examine the solubility of formazan without medium and in medium with different solvents. The OD of the colored formazan solution, dissolved in different solubilization solvents, was measured at a wavelength of 570 nm.

Figure 1. Formazan solubility depends on cell seeding number: As described in the experimental section, the BALB/3T3 cells were seeded at densities of 2500, 5000, and 10,000 cells in 100 μL per well in separate 96 well plates. After incubated with MTT. (A) The medium was completely removed from each well and (B) Medium was retained in the wells of the plate. Subsequently, 200 μL of different solubilization solvents (DMSO, DMSO containing 0.005 N HCl, 10% SDS in water with 0.005 N HCl, 5% SDS with 0.005 N HCl in DMSO, 10% SDS with 0.005 N HCl in DMSO) was added to each well. Both plates were then incubated at 37˚C for 1 h. The absorbance of was measured at 570 nm. Wells with medium and different solubilizing solvents were used for background correction. Data represent the mean of duplicate wells, and results are shown as the average of two independent experiments.

BALB/3T3 cells were incubated with MTT in two independent 96-well plates for 4 hours at 37˚C to assess the efficiency of various solvents in dissolving formazan. In the first plate, the culture medium was carefully aspirated, leaving the cells and formazan crystals undisturbed, whereas in the second plate the medium was retained. Subsequently, 200 µL of the respective solvents was added to each well of both plates. The plates were maintained at 37˚C, and formazan dissolution was monitored at predetermined time points (5 min, 10 min, 30 min, 1, 2, 3, 4, 5, and 24 h). The most effective solvent for dissolving formazan was determined by comparing DMSO, DMSO with 0.005 N HCl, 5% SDS and 0.005 N HCl in DMSO, 10% SDS and 0.005 N HCl in DMSO, and 10% SDS and 0.005 N HCl in water, both in without and with a medium (Figure 2(A) and Figure 2(B)). The results showed that after discarding the medium, DMSO accelerated the rapid dissolution of formazan (Figure 2(A)), which is consistent with previously reported observations [10]. In the absence of culture medium, DMSO effectively dissolved formazan, resulting in a bright purple solution with high OD (Figure 2(A)). In contrast, formazan dissolution in DMSO with 0.005 N HCl was incomplete (Figure 2(A)), resulting in lower optical density compared to DMSO alone. These findings suggest that formazan is probably unstable under acidic conditions, resulting in slower degradation and reduced solubility in acidified solvents.

Figure 2.The solubility of water-insoluble formazan was evaluated using the MTT assay. Procedure as described in the experimental section. Followed by the incubation of MTT (A) Complete removal of culture medium without disturbing adherent cells or formazan crystals, and (B) Retention of culture medium in the wells. Subsequently, separately 200 μL of different solubilization solvents was added to each well: DMSO, DMSO containing 0.005 N HCl, 10% SDS in water with 0.005 N HCl, 5% SDS in DMSO with 0.005 N HCl, or 10% SDS in DMSO with 0.005 N HCl. Plates were incubated at 37˚C for 5 min to 24 h. Absorbance was measured at 570 nm at each time point. Background correction was performed using blank wells containing complete medium, while wells without medium were corrected using the corresponding solubilization solvent. All values represent the mean of duplicate wells, and data are presented as the average of three independent experiments.

Figure 2(A) demonstrates that 5% and 10% SDS in DMSO containing 0.005 N HCl exhibited similar dissolution patterns. Although these SDS-based solvents produced a purple solution, the OD was lower than that achieved with DMSO alone, indicating reduced efficiency. Furthermore, formazan dissolved more slowly and often incompletely in acidified DMSO with both 5% and 10% SDS (Figure 2(A)). These findings suggest that, while acidified SDS in DMSO is capable of dissolving formazan, the combination is suboptimal. A water-based SDS solution is therefore recommended for consistent dissolution, as acidified DMSO may promote SDS precipitation and diminish its effectiveness. To further evaluate this conclusion, formazan solubility was tested in water containing 10% SDS and 0.005 N HCl. OD measurements revealed that acidified SDS in water produced a lower OD (Figure 2(A)). These results indicate that the hydrophobic formazan crystals dissolve inefficiently in water-based SDS solutions. This reduced solubility in acidified aqueous SDS likely promotes the formation of precipitates or leaves crystals undissolved. Collectively, the data demonstrate that DMSO is the most effective solvent for formazan dissolution, particularly in the MTT assay when the culture medium is completely removed prior to solvent addition.

Similarly, formazan was dissolved by adding various solubilizing solvents directly to wells containing culture medium, including DMSO, DMSO with 0.005 N HCl, 5% SDS with 0.005 N HCl in DMSO, 10% SDS with 0.005 N HCl in DMSO, and 10% SDS with 0.005 N HCl in water. Among these, DMSO supplemented with 10% SDS and 0.005 N HCl produced the highest OD (Figure 2(B)), indicating that this combination most efficiently and rapidly solubilizes formazan crystals and cells. Collectively, these findings demonstrate that 10% SDS in DMSO with 0.005 N HCl is the optimal solvent for rapid formazan dissolution when the culture medium is retained. However, implementing this mixture of solvent has larger benefits since it may be introduced directly to formazan crystals in a medium and does not require overnight incubation [14]. Similarly, the results demonstrated that solvents such as 5% SDS with 0.005 N HCl in DMSO, DMSO containing 0.005 N HCl, and DMSO alone dissolved formazan crystals more slowly. Moreover, the OD values obtained with these solvents were lower than those measured with 10% SDS and 0.005 N HCl in DMSO, indicating that formazan was not fully solubilized under these conditions.

Complete dissolution of water insoluble formazan crystals in culture medium typically requires a solubilization solution containing high concentrations of SDS [9], most commonly at 10%. The OD increases proportionally with the degree of formazan dissolution, indicating the efficiency of the solubilization process. Furthermore, 10% SDS with 0.005 N HCl in water was evaluated, and this condition produced the lowest OD, indicating that formazan crystals were least soluble in this aqueous SDS solution when culture medium was present. These findings show that, when the culture medium is present during the MTT experiment, 5% SDS with 0.005 N HCl in DMSO, 10% SDS with 0.005 N HCl in DMSO, or DMSO alone are the most effective solvents for dissolving formazan when compared to 10% SDS and 0.005 N HCl in water solution. Furthermore, because the media did not need to be removed, the modified MTT assay can be used on adherent cells both with and without medium. It is also likely applicable to suspension types of cells. The mean of three separate experiments is used to report all results.

The results suggest that incubation of 5000 BALB/3T3 cells per well in a 96-well plate with 0.5 mg/mL MTT for four hours gave the optimal level of MTT reduction. However, both cell density and MTT concentration were observed to influence the saturation time point [6]. The results demonstrated that wells in which the medium was retained yielded the highest OD values, whereas wells with medium removal showed the lowest OD. This difference can be attributed to the contribution of the medium itself to the absorbance signal, thereby elevating the total OD. Moreover, once formazan is dissolved in DMSO or other DMSO-based solvents, the OD values remain stable for several hours. Overall, the results show that formazan dissolves quickly in the presence of medium when a mixture of 10% SDS and 0.005 N HCl in DMSO is used, while formazan dissolves effectively in DMSO alone after the medium has been completely removed. In addition, this result can be related to the chemical properties of DMSO, a polar organosulfur molecule that is miscible with water and a variety of organic solvents, allowing it to dissolve polar and nonpolar chemical compounds.

Some medium components can reduce MTT, leading to misleading results in formazan solubility assays. To mitigate this, blanks containing the complete medium, but no cells were prepared and analyzed in parallel with experimental samples. In the absence of metabolically active cells, medium components such as FBS, amino acids, reducing sugars, vitamins, and other reducing agents may react with MTT to generate formazan crystals. This abiotic reduction produces artificially elevated OD values, resulting in inaccurate results. Therefore, the possibility of non-enzymatic reduction of MTT by culture medium containing FBS and various solubilizing solvents was examined in two independent 96-well plates. Found that none of the five tested solvents such as DMSO, DMSO with 0.005 N HCl, 10% SDS in water with 0.005 N HCl, 5% SDS with 0.005 N HCl in DMSO, or 10% SDS with 0.005 N HCl in DMSO were capable of reducing MTT to formazan (Figure 3(A)). In all cases, the OD values obtained from the solvent mixtures were nearly identical to those of the blank control, confirming the absence of MTT reduction. These results clearly indicate that solvent-based solubilizing mixtures do not contribute to non-enzymatic MTT reduction.

Figure 3. Non-enzymatic reduction of MTT by culture medium components and solubilizing solvents was assessed using cell-free controls, as described in the experimental section. Two sets of controls were prepared: (A) 100 µL of each formazan-solubilizing solvent incubated with MTT, and (B) 100 µL of culture medium incubated with MTT. Following incubation, 200 µL of the respective solubilization solvent (DMSO, DMSO containing 0.005 N HCl, 10% SDS in water with 0.005 N HCl, 5% SDS in DMSO with 0.005 N HCl, or 10% SDS in DMSO with 0.005 N HCl) was added to each well. Plates were further incubated at 37˚C for 5 min to 24 h. Then absorbance was measured at 570 nm at each time point. Data represent the mean of duplicate wells, and results are presented as the average of two independent experiments.

When different solvents were individually added to the culture medium, the plate containing medium (Figure 3(B)) revealed that 10% SDS with 0.005 N HCl in DMSO produced the highest OD, followed by 5% SDS with 0.005 N HCl in DMSO. DMSO alone and DMSO with 0.005 N HCl also generated measurable OD values, though these were lower than those obtained with SDS-containing solvents. These results suggest that components of the medium may contribute to non-enzymatic reduction of MTT, while 10% SDS with 0.005 N HCl in DMSO provides the most efficient formazan solubilization, yielding the highest OD. In comparison, 5% SDS with 0.005 N HCl in DMSO yielded lower OD values, suggesting that a higher SDS concentration (10%) is required for complete dissolution of formazan in medium. The markedly lower OD values observed with DMSO and DMSO containing 0.005 N HCl further emphasize the essential role of SDS in effective solubilization. The lowest OD was recorded with 10% SDS in water containing 0.005 N HCl (Figure 3(B)), confirming that dissolution of water-insoluble formazan requires the presence of an organic solvent such as DMSO in combination with SDS for complete solubilization.

The concentration of HCl used in formazan solutions varies across protocols. Commonly reported values include 0.01 M, 0.025 M [9]-[14], and 0.04 N HCl [21]. In assays such as MTT, formazan crystals are dissolved in these acidic solutions, and both the stability and solubility of the resulting solution can be affected by the specific HCl concentration employed. The influence of HCl concentration in formazan‑solubilizing solvents was examined using 0.01 N HCl to minimize interference from phenol red in the growth medium. Because the acid converted phenol red to yellow, its contribution to OD at the measured wavelength was negligible. After incubation, the culture medium was carefully removed without disturbing the cells and formazan crystals (Figure 4(A)) or medium retained in the wells (Figure 4(B)). Formazan crystals were dissolved in five different solvents: DMSO, DMSO with 0.01 N HCl, 10% SDS in water with 0.01 N HCl, 5% SDS in DMSO with 0.01 N HCl, and 10% SDS in DMSO with 0.01 N HCl. Compared with formazan dissolved in DMSO, DMSO with 0.005 N HCl, 10% SDS in water with 0.005 N HCl, 5% SDS in DMSO with 0.005 N HCl, and 10% SDS in DMSO with 0.005 N HCl (Figure 2(A) and Figure 2(B)), the OD values obtained with the higher‑acid solvents were consistently lower.

Formazan, the chromogenic product of the MTT assay, displays a characteristic purple color when fully solubilized. However, at highly acidic pH levels, formazan becomes unstable and may degrade or undergo shifts in its absorption spectrum, leading to yellow discoloration or complete loss of color. These spectral changes reduce absorbance at the standard quantification wavelength (570 nm), thereby lowering OD values even when the actual amount of formazan generated by the cells remains constant. Consequently, the concentration of HCl in the solvent system is a critical parameter, as excessive acidity disrupts the colorimetric properties of formazan and can result in underestimation of cell viability or metabolic activity. In this study, SDS‑ or DMSO‑based solvents containing 0.005 N HCl produced higher OD values (Figure 2(A) and Figure 2(B)), whereas increasing the HCl concentration to 0.01 N resulted in reduced OD values (Figure 4(A) and Figure 4(B)). These findings demonstrate the importance of optimizing HCl concentration to ensure reliable and reproducible outcomes in formazan‑based assays.

Figure 4.The effect of HCl concentration in formazan-solubilizing solvents was evaluated by monitoring changes in optical density. As described in experimental section after incubated with MTT. (A) complete removal of culture medium without disturbing adherent cells or formazan crystals, and (B) Retention of culture medium in the wells. Subsequently, 200 µL of one of the following solubilization solvents was added to each well: DMSO, DMSO containing 0.01 N HCl, 10% SDS in water with 0.01 N HCl, 5% SDS in DMSO with 0.01 N HCl, or 10% SDS in DMSO with 0.01 N HCl. Plates were incubated at 37˚C for periods ranging from 10 min to 24 h. Then absorbance was measured at 570 nm at each time point. Background correction was performed using wells containing complete medium and the respective solvents. Data represent the mean of duplicate wells, and results are presented as the average of two independent experiments.