Glucose metabolism is critical for Trypanosoma brucei, yet little is known about how the cells sense and respond to changing glucose levels in the environment. We have developed parasites expressing a pH biosensor that allows us to monitor glycosomal pH in live parasites, which will aid in the understanding of parasites'dynamic responses to nutrient fluctuation. A challenge for this experiment would be the availability of a flow cytometer equipped with a microtiter plate reader, as well as the appropriate filter sets to measure biosensor fluorescence.
Additionally, T.brucei is a risk group two organism, so the cytometer must be housed within a biosafety level two facility. It has been discovered that the pH level of glycosomes can affect glycolysis as hexokinase and phosphofructokinase are pH sensitive. However, the pH probes that were previously used did not accurately target to the glycosome in the bloodstream form.
A better tool was needed to study the relationship between glycosomal pH and glycolysis in this parasite life stage. The pHluorin2-PTS1 Sensor is a tool that is better at localizing to the glycosome than chemical pH probes in this life stage. It's user friendly, as you only need to induce expression of the pH sensor overnight and the cells will make and localize pHluorin2 to the glycosome.
Using flow cytometry to measure pHluorin2 increases throughput and improve statistical measurements compared to microscopy. To begin, take pHluorin-expressing Trypanosoma brucei cells, add tetracycline or doxycycline and incubate overnight to induce pHluorin expression. Then transfer three milliliters of each culture into separate 15-milliliter tubes.
Then centrifuge the cells at room temperature for 10 minutes at 1000 times G.Remove the supernatant and resuspend the cells in one milliliter of PBS. After centrifuging the cells for the fourth time, remove the supernatant and resuspend the cell pellet in PBS with varying concentrations of glucose. Add either propidium iodide or thiazole red to distinguish dead from live cells.
Then transfer each sample to 5-milliliter tubes compatible with the flow cytometer. To begin, start the flow cytometer software. Open a new experiment.
Add the desired number of samples and name them. Then set up a YL2-H or RL1-H channel histogram, a forward scatter area versus side scatter area dot plot, a forward scatter area versus forward scatter height plot, and a BL1-H versus VL2-H channel plot. Place the unstained wild type Trypanosoma brucei control cells onto the sample injection port, and adjust the stage position.
To decrease the file size, uncheck the channels that are not intended to be recorded. Start at a low flow rate to allow for adjustments, then click Run to begin data acquisition. Fine tune the YL2 or RL1 voltage to align the main peak within 10 to the third to 10 to the fourth relative fluorescence intensity units.
Adjust forward scatter and side scatter voltages to ensure more than 90%of events are within the dot plot range. Set the forward scatter threshold to exclude debris without omitting viable cells. Modify VL2 and BL1 channel settings to position the primary peak of the unstained wild type control within 10 to the two to 10 to the fourth relative fluorescence intensity units.
Click Record and measure at least 10, 000 events. Then place the first-induced pHluorin-expressing Trypanosoma sample, stained with either propidium iodide or thiazole red, onto the sample injection port. Again, start data acquisition at a low flow rate after performing the rinse.
Monitor each plot to ensure that more than 90%of events are correctly captured, and that the VL2-H and BL1-H channels are not saturated. Save the data from each sample in FCS file format and prepare for analysis. To begin, perform flow cytometry on pHluorin-expressing Trypanosoma brucei cells.
Open a new layout and drag and drop the acquired fcs files into the layout window. For gating live cells, select the unstained wild type control to open a window. Analyze the data using histograms on either the YL2-H or RL2-H channel.
In this case, use a YL2-H histogram since samples were stained with propidium iodide. Next, establish a bisector gate to separate live and dead cells, labeling the left gate as live and the right gate as dead. Apply and adjust this gate across all samples.
Ensure the gate is properly drawn by viewing the gate on multiple samples in the dataset. On the unstained wild type control, double-click on the live gate. Set the dot plot's X-axis to forward scatter area and Y-axis to side scatter area.
Then use Polygon Gate tool to outline the cell population, labeling it cells. Exclude debris or dying cells and aggregates by being cautious about their typical positions in the dot plot. Apply the cells gate under the live gate for all samples, ensuring it covers the likely cell population for all samples.
Double-click on the cells gate on the unstained wild type control to view the events within this gate. To adjust the dot plot, set the X-axis to forward scatter area and Y-axis to forward scatter height. Identify the diagonal distribution of single cells on this dot plot, distinguishing them from doublets.
Utilize the Polygon Gate tool to encircle the singlet events, naming this gate singlets. Apply the singlets gate under the cells gate for all samples, ensuring it excludes doublets while including singlets. And adjust the gate as necessary across different samples.
On the unstained wild type control sample, double-click on the singlets gate. Alter the dot plot's X-axis to BL1-H and the Y-axis to VL2-H. Using the Polygon Gate tool, draw a diagonal gate to capture pHluorin2 fluorescent cells in VL2 and BL1.
Label this gate as pHL+Apply the pHL+gate under the singlets gate for all samples. Adjust the pHL gate to cover cells with higher fluorescence intensity than the autofluorescent cells in the wild type control. To export the statistics, click on the table editor, then Edit bar, and finally select Add Column.
Incorporate columns for total ungated count, pHL+count, live frequency of total, pHL+frequency of parent, pHL+median VL2-H, and pHL+median BL1-H. To adjust export settings in the table editor, set display to To File and text to CSV. Select the file destination and name, then click Create Table.
A gradual, mild acidification was observed over time in response to starvation, which plateaued by 90 minutes. After reintroducing glucose, as indicated by the green line, glycosomal pH returned to pre-starvation levels in 30 minutes, suggesting that glycosomal pH is dynamic and regulatable in response to glucose.
