This method can help us to answer key questions in the immuno-metabolism field. The advantage of this technique is that it allows the quantification of mitochondria or lysosomes in individual living cells, in a complex mixture of different cell types. This method for studying T and B cells can also be applied to many other cell types such as macrophages, dendritic cells, or any primary cells harvested from a tissue sample.
The procedure will be demonstrated by Chin-Wen Wei, a graduate student from my laboratory. To label the organelles for quantification, first, add one times ten to the sixth mouse T cells to one round bottom fax tube per marker. And pellet the cells by centrifugation.
pre-warmed to 37 degrees Celsius serum-free culture medium to dilute the organelle specific probe stock solutions to final working concentrations in the medium. And re-suspend the cells in 100 microliters of probe dye per tube. Next, incubate the cells in a cell culture incubator for the appropriate staining period, stopping the reaction at the end of the incubation with one milliliter of ice cold fax buffer per tube.
Then, collect the cells with another centrifugation and re-suspend the pellets in 100 microliters of the pre-titrated 24G2 hybridoma supernatant on ice. After ten minutes, add one milliliter of fax buffer per tube. Collect the cells by centrifugation and label the cells with 50 microliters of the pre-titrated fluorescence conjugated antibody cocktail of interest.
Add the appropriate concentration according to the table. And fax buffer for 20 minutes on ice, protected from light. At the end of the incubation, add one milliliter of fax buffer per tube.
Collect the cells by centrifugation. And re-suspend the cells in 300 microliters of fax buffer supplemented with one microgram per milliliter of propidium iodide. Immediately after adding the nuclear and chromosome counterstain, turn on the flow cytometer analysis computer, flow cytometer, fax acquisition software, and any other accessory devices depending on the machine platform.
Run PBS through the system for about one minute to ensure that the flow line is filled with PBS and sheathe buffer. Open a new experiment and set the cytometer parameters according to the manufacturer's instructions. Filter the cells through a 70 micrometer cell strainer.
And vortex prior to the acquisition of each sample to avoid clumps and doublet formation. Open the Auto Compensation setting, and create dot plots for each fluorescent parameter. After all of the voltages have been set, adjust the compensation and apply the compensation to the samples.
Create a forward scatter versus side scatter plot and optimize the voltages so the cells of interest appear within the plot. Create a forward scatter versus forward scatter height plot and gate the single cells. Create a forward scatter area versus side scatter area plot and gate the lymphocytes.
Create a forward scatter area versus propidium iodide plot and gate the live cells. After all of the voltages have been set, adjust the compensation and apply the compensation to the samples. Then load the first sample tube, create the appropriate cell surface marker plots, and collect at least 1, 000 events of the population of interest.
When all of the samples have been run, export the flow data for subsequent analysis. And save the experiment as a template to preserve the cytometer settings and parameters for similar experiments in the future. Final site mitochondrial contents are the lowest in double negative one T cells, peak at double negative stage two and three, and slightly decrease in double negative stage four.
With double positive thymocytes demonstrating higher numbers of mitochondria than CD4 and CD8 single positive thymocytes, suggesting that the mitochondrial contents fluctuate during development. CD4 and CD8 single positive thymocytes exhibit a higher mitochondrial mean fluorescence intensity than splenic CD4 or CD8 T cells, suggesting that naive T cells that recirculate in the oxygen rich blood environment have an even lower mitochondrial mass than thymocytes. Interestingly, this reduction of mitochondrial contents is more prominent in the CD8 than the CD4 T cell lineage, and the T cell activation further decreases the presence of these organelles.
Relatively low, but detectable, lysosomal contents are observed in all thymocyte populations, with a more prominent presence in double negative one thymocytes. In the periphery, a relatively high number of lysosomes are detected in memory and effector CD8 positive T cells. Combining organelle specific dyes and surface marker with flow cytometry is a powerful way to characterize the metabolic status of cells in a complex mixture.
Additional organelle specific dye can be used to detect endoplasmic reticulum, autophagic vacuole, or other intracellular compartment of interest.
