Measurement: Background

This year, our team aimed to develop Afamin-Wnt3a conditioned cultured media (CM) as a potential substitute for fetal bovine serum (FBS). Throughout our Engineering Cycle (please see our Engineering page for more information, we came across two major quantitative experiments requiring categorical measurements:

• Bradford Assay: Confirming the molecular viability of the engineered HEK293 cells (Part: BBa K4869003) co-transfected with the BII-CMV-AfmW3A Vector (Part: BBa K4869000) and Super PiggyBac Transposase Expression Vector (Part: BBa K4869001).
• Prestoblue Assay (Cell Proliferation Assay): Assessing the effect of Afamin-Wnt3a conditioned CM on bovine myoblast cell (MyoB) proliferation.

Bradford assay was critical to the ‘Build’ phase of our Engineering Cycle, whereas Prestoblue assay was crucial to the ‘Test’ phase. However, we faced two major limitations. First, the Bradford assay requires a normalization reference (i.e., standard curve) to quantify the total protein concentration accurately. Second, the Prestoblue assay also requires a standard curve to quantify the metabolic activity of the living cells, which must be reliably and empirically presented as the rate of cell proliferation. Accordingly, our objectives for this year’s measurement protocols were the following:

• Bradford Assay: Obtaining a standard curve that could reliably yield the total protein concentration of the engineered HEK293 cells containing Wnt3a and Afamin genes.
• Prestoblue Assay (Cell Proliferation Assay): Obtaining a standard curve that could reliably yield metabolic activity of living MyoB cells is a proxy for the number of cells and the rate of cell proliferation.

Measurement Part 1: Bradford Assay

Measurement Background

The Bradford assay, developed by Marion M. Bradford in 1976, is a widely used biochemical assay for quantifying protein concentration in a solution. The reaction is highly dependent on the composition of amino acids of the targeted proteins. A colorimetric assay relies on a dye's color change in response to protein binding.

Measurement Principle

The Bradford assay is based on the principle that the Coomassie Brilliant Blue G-250 dye binds to proteins in an acidic solution. When the dye binds to proteins, it shifts from brown to blue. The intensity of this color change is directly proportional to the protein concentration in the sample. The primary reagent in the Bradford assay is Coomassie Brilliant Blue G-250 dye dissolved in an acidic solution (usually phosphoric acid or sulfuric acid). The dye interacts with positively charged amino acid residues on the surface of proteins, causing the solution to change color.

Measurement Protocols

[Materials]

• Protein samples
• Bovine serum albumin (BSA) standard solution (2 mg/mL)
• Bradford reagent
• Clear, flat-bottom 96-well microplate
• Microplate reader
• Pipettes and tips
• Distilled water

[Procedures]

1. The following condition was used to prepare the six samples of standard curve sample (Figure 1). We diluted 2 mg/mL BSA via distilled water to obtain six standard samples with BSA concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 µg/µL, respectively.
2. Add 5 µL of standard samples to the well in the 96-well microplate.
3. Add 95 µL Bradford reagent to each well.
4. Mix the solution in the microplate wells gently by pipetting up and down (Figure 2).
5. Allow the standard and sample wells in the microplate to incubate at room temperature for approximately 10 minutes. During this time, the Bradford reagent will bind to the proteins.

1. Set your microplate reader to measure absorbance at 595 nm.
2. Zero the microplate reader using the blank well containing distilled water.
3. Measure the absorbance of each standard solution and your protein samples in the microplate.

1. Plot a standard curve using a spreadsheet to show the known concentrations of the BSA standard and their corresponding absorbance values.
2. Determine the absorbance of our protein samples.
3. Using the standard curve, find the corresponding protein concentration for each sample based on their absorbance values (Figure 3).

The quantification process employed the trendline equation derived from the BSA standard curve. This equation directly correlates the absorbance values obtained from our samples and the corresponding protein concentrations. We determined the protein concentrations for each condition examined in our experiment using this established relationship. Using the equation of the standard curve in Figure 3, the total protein concentrations were calculated:

• Absorbance (595 nm) = 0.834 x Total Protein concentration (µg/µL) + 0.286
• Total Protein concentration (µg/µL) = [Absorbance (595 nm) - 0.286] / 0.834

The outcomes of this quantification provided the following total protein concentrations for the respective cell culture media conditions:

• Negative control HEK293: 0.81 µg/mL
• 1:2.5 transfected HEK293: 0.79 µg/mL
• 1:5 transfected HEK293: 0.75 µg/mL

Measurement Discussions

Before the Bradford assay, we lacked normalization reference for Wnt3a and Afamin. However, utilizing BSA as an alternative normalization reference, we obtained the least-squares regression line predicting absorbance at 595 nm for each total protein concentration. The regression line represented a strong positive linear correlation (r=0.99), validating the reliability of predicting total protein concentration from the measured spectrophotometric absorbance (Figure 3). We thereby were able to quantify the total protein concentration of the engineered HEK293 cells containing both Afamin and Wnt3a genes, validating the molecular integrity following co-transfection by the PiggyBac Vector System. Here, we described the process of (1) obtaining a BSA standard curve and (2) using the regression line to predict the total protein concentration in HEK293 cells (Figures 1~3).

To obtain a calibrated regression line, we employed a negative control: BSA solution with a concentration of 0 µg/mL. The unit of the x-axis was µg/µL, and the unit of the y-axis was arbitrary units (a. u.). The explanatory and response variables were appropriately measured and presented as a least-squares regression line with a strong positive linear association. We highly recommend future iGEM teams use such control/calibration measures to obtain correct data. This measurement technique may be helpful for teams lacking the proper normalization reference when quantifying the total protein concentration. Utilizing our approach, however, may be challenging to obtain the relative abundance, or molar ratio, between more than one protein. We also could not determine the relative abundance between Afamin and Wnt3a proteins. It also must be acknowledged that such prediction may hold valid if two distinct samples (in our case, BSA and the engineered HEK293 cells) are similar regarding amino acid composition and size.

Measurement Part 2: Prestoblue Assay

Measurement Background

The PrestoBlue assay, a highly versatile and widely adopted technique in cell biology, is a crucial tool for assessing cell proliferation with precision and efficiency. This assay relies on quantifying the metabolic activity of cells, thereby offering a reliable means of estimating cell numbers.

Measurement Principle

The PrestoBlue assay is grounded in the principle of measuring the metabolic activity of cells as a proxy for cell proliferation. It accomplishes this by utilizing a colorless resazurin dye, which is non-toxic to cells. When added to cell culture, this dye is taken up by living cells and reduced by mitochondrial enzymes within metabolically active cells. This reduction converts the colorless resazurin into a highly fluorescent and colorful compound known as resorufin. The intensity of this fluorescence or absorbance is directly proportional to the number of metabolically active cells present in the culture. Thus, the assay provides a quantitative measure of cell proliferation by assessing the rate at which resazurin is transformed into resorufin, effectively reflecting changes in cell numbers over time.

Measurement Protocols

[Materials]

• MyoB cultured cells
• PrestoBlue reagent (commercially available)
• 96-well microplate
• Microplate reader
• Pipettes and tips
• Sterile cell culture hood or laminar flow hood
• CO2 Incubator

[Procedures]

To prepare the cell samples, 96-well plates were used, and 1x103 MyoB cells were added to each well for the experiment. Additionally, standard curve cells were prepared by varying the number of cells to 0, 1x103, 2x103, 5x103, 10x103, and 15x103. All samples were prepared for the duplicate measurements.

• Aspirate the culture medium from the wells containing MyoB cells.
• Rinse the cells once with phosphate-buffered saline to remove any residual medium.
• Add the 90 µL of treatment (Without FBS, With 10% FBS, With 10% Afamin-Wnt3a CM, With 20% Afamin-Wnt3a CM) on each appropriate position of the 96 well plate.
• Incubate the cells for 0, 24, 48,72, and 96 hours of the treatment.
• For each time point, PrestoBlue solution was added to the sample (10 µL) to each well-containing cell (final concentration should not exceed 10%).
• Incubate the cells with the PrestoBlue reagent at 37°C for 30 minutes.

• Following incubation, a microplate reader was used to measure the absorbance at 570 nm (with a reference wavelength of 600 nm). Record the absorbance values for each well.
• Calculate cell viability by comparing the absorbance values of the treated cells with those of the untreated control cells.

The PrestoBlue assay showed a strong correlation (R²=0.991) between cell number and absorbance values, indicating accurate and consistent results. (Figure 4). This correlation proves that the PrestoBlue assay is a reliable method for quantifying the changes in live cell numbers. Additionally, this high correlation demonstrates the success of our optimized experimental conditions for the PrestoBlue assay.

Using the absorbance value of each sample, we calculated the number of live cells using the following equation:

• Absorbance (570/600nm) = 0.1859 x Number of cells (x103) + 0.2943

After 96 hours of incubation, we observed that the sample treated with 10% FBS had the highest mean live cell count of 19.50 x 103 cells. The second highest mean live cell count was in the sample treated with 10% Afamin-Wnt3a CM, which had 10.04 x 103 cells. The third highest mean live cell count was in the sample treated with 10% Afamin-Wnt3a CM, which had 6.54 x 103 cells. On the other hand, the sample without FBS showed a negative mean live cell count of -0.48 x 103 cells, indicating that very few live cells were measured. This result could be due to experimental error in measuring such low numbers of cells.

Measurement Discussions

Prior to the Prestoblue assay, we needed a clearly defined method to assess the effect of Afamin-Wnt3a conditioned CM on the proliferation of MyoB. Realizing that metabolic activity (as quantified by absorbance) could serve as a proxy for the number of living cells in a given sample, we first obtained a least-squares regression line (i.e., standard curve) that predicts absorbance, or the metabolic activity, from the number of living cells (Figure 4). This regression line presented a strong correlation (r=0.99), validating the use of Prestoblue assay to quantify both the number of living cells and the rate of MyoB proliferation. Consequently, we measured absorbance at various incubation periods (with varying concentrations of Afamin-Wnt3a conditioned CM, Figure 5); the regression line was used to calculate the number of living cells from the measured absorbance (Figure 6). Subsequently, a scatterplot that presents the relationship between the incubation period and the number of living cells can be obtained, allowing us to acquire the rate of cell proliferation (Figure 7). In our experiment, we used this approach to validate that Afamin-Wnt3a conditioned CM, indeed, induces MyoB proliferation.

To obtain a calibrated regression line, we employed a negative control: a well with 0 cells. The unit of the x-axis was the number of cells (x103), and the unit of the y-axis was the absorbance in arbitrary units (a.u.). The explanatory and response variables were appropriately measured and presented as a least-squares regression line with a strong positive linear association. We highly recommend future iGEM teams use such control/calibration measures to obtain correct data. As we obtained the absorbance of each sample (with varying concentrations of Afamin-Wnt3a conditioned CM), the values were converted to the number of living cells using the regression line–allowing us to present the number of living cells at various time intervals.

This measurement technique will be helpful for future iGEM teams willing to plot the rate of cell proliferation at various time intervals. This is critical for biomanufacturing in various respects of Synthetic Biology, as the rate of cell proliferation is a crucial element that must be controlled in a sophisticated manner. In order to achieve our goals this year, we provided a model or a set of relevant protocols and background information. By establishing a standard regression line and performing several calculations, we can easily and effectively determine the rate of cell proliferation by combining the Prestoblue assay with quantitative statistics. However, it is important to note that errors in measuring low cell counts may result in negative cell counts, so caution should be exercised.

References