The overall goal of this procedure is to monitor the process of trans-plasma membrane electron transport in real time through spectrophotometric assays. This method can help answer key questions in the radox biology field regarding the mechanism of trans-plasma membrane electron transport and how a cell's redox environment is maintained. The main advantage of this technique is that the assay is a real time, multi-well, quick spectrophotometric assay.
To begin, seat C2C-12 adherence cells at two times 10 to the 4th cells per square centimeter in rows A through F of a 96 well plate, then cover the cells with growth media. Incubate the cells in a 37 degree Celsius incubator with 5%carbon dioxide. After 24 hours in culture, aspirate the growth media, wash with 150 microliters of PBS, replace with differentiation medium, and incubate the cells for an additional 24 to 48 hours.
Next, prepare a stock WST-1 and PMS solutions. Make a 10 millimolar stock solution of WST-1 by dissolving 033 grams of WST-1 in five milliliters of PBS. Then, vortex the solution.
Then, prepare the stock solution of PMS by dissolving 0023 grams of PMS in 1.5 milliliters of the ionized water. Vortex the solution. To prepare the working solution, add glucose to PBS to make 11.4 milliliters of a five millimolar glucose solution.
To this solution, add 480 microliters of the WST-1 stock solution for a final concentration of 400 micromolar WST-1 and 48 microliters of the PMS stock solution for a final concentration of 20 micromolar PMS. Vortex the solutions to mix. Then divide the solution into two six milliliter aliquots.
Depending on the assay, add the relevant components described here. With the solutions now prepared, aspirate the differentiation media from the cells and wash them once with 150 microliters of PBS. Remove the PBS and add 100 microliters from aliquot one into columns one through six.
And add 100 microliters from aliquot two to columns seven through 12 of the 96 well plate. Place the 96 well plate into a plate reader and measure the absorbance values every 10 minutes for one hour at 438 nanometers. After the final reading, aspirate the media and wash each well with 150 microliters of PBS.
Then, aspirate the PBS. For analysis, calculate the change in absorbance for each well by subtracting the initial absorbance for the well from the absorbance at any later time point for that same well. Correct this change based on the change in absorbance that is observed in the background wells.
Once background corrected, normalize the absorbance data to the 60 minute control measurements. Alternatively, normalize by the protein content in each well using a standard BCA protein assay. Finally, quantify the data to obtain the amount of WST-1 reduction by microgram of protein.
To begin the DPIP reduction assay, grow and differentiate C2C-12 adherence cells, as shown for the WST-1 reduction assay. Next, prepare a stock solution containing 10 millimolar DPIP by dissolving 029 grams of DPIP in 10 milliliters of the ionized water. Then vortex the solution.
Confirm the concentration by measuring the solution's absorbance at 600 nanometers using a spectrophotometer. To make the working solution, add 120 microliters of the 10 millimolar DPIP stock solution to 11.88 milliliters of a five millimolar solution of glucose and PBS for a final DPIP working concentration of 100 micromolar. Then, vortex the solution.
Then divide the solution into two six milliliters aliquots. Depending on the assay, add the relevant components described here. With the solutions now prepared, aspirate the differentiation media from the cells and wash them once with 150 microliters of PBS.
Remove the PBS and add 100 microliters from aliquot one into columns one through six, and 100 microliters from aliquot two to columns seven through 12 of the 96 well plate. Place the 96 well plate into a plate reader and measure the absorbance values every 10 minutes for one hour at 600 nanometers and analyze the resulting values as described in the WST-1 reduction assay analysis. To begin, prepare two solutions.
First, prepare six milliliters of 400 micromolar WST-1 and 20 micromolar PMS in PBS. Then prepare six milliliters of a 100 micromolar solution of DPIP and PBS. Prepare the first 96 well plate by adding 100 microliters of a WST-1/PMS solution to rows A through D in a flat bottom plate in the absence of cells, and measure the absorbance in a spectrophotometer at 438 nanometers.
Then prepare a second 96 well plate by adding 100 microliters of the DPIP solution to rows A through D in a flat bottom plate in the absence of cells, and measure the absorbance in the spectrophotometer at 600 nanometers. Next, add one microliter of 10 millimolar ascorbate to half of the wells for a final concentration of 100 micromolar. Monitor the absorbance in the plate until it stabilizes.
After this, repeat with DPIP. Upon stabilization, determine whether the produced electron acceptors are substrates. For ascorbate oxidase for super oxide dismutates, by adding either ascorbate oxidase or super oxide dismutates to each well and monitoring the absorbance change for one hour.
To monitor transplasma membrane electron transport, ascorbate oxidase was used to block the effects of ascorbate mediated reduction and superoxide dismutase was used to block superoxide mediated reduction to determine which portion of WST-1 reduction was due to each component. With the addition of ascorbate oxidase, WST-1 reduction was suppressed by about 30%indicating that about 30%of the transplasma membrane electron transport was due to the export of ascorbate. With the addition of superoxide dismutase, WST-1 reduction was suppressed by about 70%indicating that about 70%of the transplasma membrane electron transport was due to extracellular superoxide release.
To determine if reduced DPIP is a substrate for ascorbate oxidase or super oxide dismutase, DPIP levels were reduced with ascorbic acid. After 20 minutes, ascorbate oxidase was added, and the absorbance was monitored. The recovery shows that reduced DPIP is a substrate for ascorbate oxidase.
When super oxide dismutase was added, DPIP was also reoxidized, albeit at a slower rate;therefore, reduced DPIP is a substrate for these enzymes and not an appropriate extracellular electron acceptor to be utilized with these enzymes. Once mastered, this technique can be completed in two hours if it is performed properly. While attempting this procedure, it's important to remember to make all reagents fresh each day.
After its development, this technique paved the way for researchers in the field of redox biology to explore transplasma membrane electron transport in a variety of cell types. After watching this video, you should have a good understanding of how to measure transplasma membrane electron transport utilizing extracellular electron acceptors, as well as how to determine if the electron acceptor might be a substrate for enzymes utilized in the assay.
