The overall goal of this procedure is to determine the Lipid Droplet Index of cells cultured under different nutritional conditions. This method can help answer key questions in the Lipid Droplet and Lipid Field, such as the dynamic of accumulation and the different nutritional conditions. The main advantage of this technique is that multiple samples can be read simultaneously, because it can be implemented in the microplate.
With the start of this method when we observe refractance of Ustilago maydis cells cultured under nitrogen starvation and then realize that this might mean that these instructors might be lipid droplets. So we contacted Monica Montero from Universidade Federale do Rio de Janeiro in Brazil who implemented the Liquid Florescence Recovery Assay of lipid droplets in Saccharomyces cerevisiae to start a collaboration to adapt the procedure for Ustilago maydis. The first step in this protocol is the preparation of the required buffers and solutions.
To prepare a 10 millimolar BO-DP stock solution, dissolve 10 milligrams of BO-DP 493-503 and 3.8 milliliters of dimethyl sulphoxide. Divide into 100 microliter aliquots. Then keep in the dark a minus 70 degrees Celsius.
Prepare a five micromolar BO-DP quenching solution for the liquid florescence recover, or LFR-Assay by adding one microliter of 10 millimolar BO-DP 493-503 to two milliliters of 500 millimolar quenching solution. Grow the umeda cells for this study by starting a culture with an initial optical density of 600 nanometers of 05. Incubate the cells at 28 degrees Celsius and 180 RPM for 24 hours.
At selected times, withdraw an aliquot of the cells. During the first eight hours, withdraw five milliliters every two hours. After eight hours of growth aliquots of two milliliters are enough.
Measure the optical density at 600 nanometers of each aliquot. Centrifuge the cell suspension at 14, 000 times G for one minute at four degrees Celsius. And, discard the supernatant.
Suspend the cell pellet in one milliliter of fixing solution containing 3.7%formaldehyde in PBS. Incubate the cells at room temperature for 15 minutes. After 15 minutes, centrifuge the cell suspension at 14, 000 times G and four degrees Celsius for one minute.
Discard the supernatant. Wash the cell pellet with one milliliter of distilled water. Centrifuge the cell suspension for one minute.
Discard the supernatant and add one milliliter of distilled water for a second wash. Centrifuge again. Remove the supernatant from the second wash.
Using distilled water, adjust the optical density of the cell pellet at 600 nanometers to five, which is equivalent to 1.5 times 10 to the eighth cells per milliliter. Keep the samples at four degrees Celsius until use. Begin this procedure by turning on the spectrophotometer.
Open the SkanIt software. Click on new session. From the session tree select protocol enter the settings for the florescence wavelength and choose automatic photo multiplayer gain.
For the excitation bandwidth select 12 nanometers. In the optics, select top for excitation of the sample from the top of the well. Enter 300 RPM and low for a gentle and continuous agitation.
Select run plate out, pause, and until the user action, repeat the protocol five times. Click save and in the session name field, write a name for the session. The protocol is now ready for sample analysis.
Add 200 microliters of the 5 micromolar BO-DP quenching solution to each well of a black 96 well clear-bottomed plate. Place the plate inside the spectrophotometer chamber and incubate at 30 degrees Celsius for five minutes. From here on, protect the plate from light as much as possible.
Click the start button and read the florescence and the optical density at 600 nanometers, corresponding to the blanks. During the pause time, add five microliters of the formaldehyde fixed cell suspension to the wells and mix the samples carefully with a pipette. Put the plate back inside the spectrophotometer, and click continue.
Repeat three successive additions of five microliters of cell suspension. Mix with a pipette to make sure the cells do not precipitate before continuing with the read. In this demonstration, Excel software is used to calculate and plot a graph of florescence and absorbance against the volume of each successive addition for each sample in the microplate.
Enter the volume, florescence and absorbance data in the data sheet. Then select the whole data with the cursor, click insert and X Y scatter. Select the points for each line in the graph and insert the respective trend line with the show equation box selected.
Write down the values of the slopes of the two straight lines according to the equation. To get the lipid droplet, or LD index, divide the slope of the fluorescence line by the slope of the optical density line. To analyze the quality of the data, calculate the correlation coefficient of each straight line.
Click on the function wizard and choose coral. The readings are reliable if R is equal to or greater than 0.9. It is important to corroborate the sensitivity of this method by confocal microscope.
When cells were cultured in YPD Medium, there was an increase in the LD index during the exponential phase followed by a decline in the stationary phase. In contrast cells grown under nitrogen starvation showed a steady increase in LD index in both the exponential and stationary phases. Under nitrogen starvation, the increase in the LD index was inhibited by Soraphen A, a specific inhibitor of the Acetyl-CoA carboxylase enzyme, essential for the synthesis of fatty acids, contained in the triacylglycerols stored in lipid droplets.
Since both cultures were started with the same optical density, the results show the capacity of the assay to detect small changes in the lipid content. The sensitivity of this method was corroborated by confocal microscopy. YPD cells contain many small LDs throughout the whole cell, while nitrogen starvation produced large LDs, typically four to five per cell.
To further investigate the mobilization of LDs in U.Maydis, cells grown under nitrogen starvation for 72 hours were transferred to YPD medium. As shown, the LD index decreased following an exponential function. Since the cells were able to mobilize the LDs accumulated during nitrogen starvation, the result suggests a reduction in the triacylglycerol content.
Once mastered, this technique can be done in one hour if it is performed properly. When attempting this procedure, it's important to remember to protect the cells from light and to mix between additions. Following this procedure, other methods for example triacylglycerol or total lipid content determination can be performed to answer additional questions, like the exact lipid content in the cells.
After its development, this technique paved the way for researchers in the field of lipid biotechnology to explore new or alleged organisms in order to increase the production of biofuels or lipids in pharmaceutical applications. Even malignant cancer cells can be studied with this technique to explore the participation of lipid metabolism in disease development. After watching this video, you too have a good understanding of how to determine the lipid droplet index of these cells.
Don't forget that working with formaldehyde can be extremely hazardous and precautions such as wearing gloves should always be taken while performing this procedure.
