The goal of this project was to develop and optimize a protocol for measuring changes in the mitochondrial membrane potential (Δψm) in human leukocytes. This may serve as an early biomarker for sepsis, the leading cause of death in hospitals - particularly among trauma patients. Following trauma, patients may experience an immune response that suppresses leukocytes, increasing their susceptibility of developing an infection. There is much evidence suggesting that mitochondrial energy production in immune cells is impaired following trauma, and this can be indirectly measured by Δψm. We hypothesize that changes to the Δψm in leukocytes can serve as an early biomarker to predict an increase in risk for sepsis. To test this, the team used a system which measures changes in the Δψm with a reactive fluorescent dye. We also optimized the protocol in areas including cell collection, establishing controls, and ensuring cell viability.

For each experiment, 5 mL of blood was collected from healthy volunteer donors and the leukocytes were extracted using density gradient centrifugation. Initially, the leukocytes were then spun down; the supernatant removed; and the pellet resuspended for counting and plating. However, due to the density gradient, the team found that a wash step was needed to improve pelleting. Additionally, we found that using a swing-bucket centrifuge instead of a fixed-bucket centrifuge drastically improved pelleting and recovery.

Next, the team moved to optimize cell viability. Initially, cell count reads were low, averaging around 3 x 106 cells. We surmised that this was likely due to debris and two steps were added: an extra wash step in EasySep (PBS + 2% FBS) and another wash to remove the FBS. These changes increased cell counts and viability.

Changes to the Δψm were detected using the MITO-ID Membrane Potential Cytotoxicity kit (Enzo Life Sciences) and the resulting fluorescence was analyzed with a plate reader (Biotek Synergy). However, the plate reader was unable to read the plate at exactly 490 and 590 nm (the recommended settings). This created a high level of background and overlap which made it difficult to discern the fluorescence changes. The team switched to using flow cytometry for this purpose and were able to more precisely determine the change in Δψm.

To ensure the validity of the data, the team then focused on establishing the positive and negative controls. For the negative control, we used CCCP to destroy the Δψm. Initially, we tested different amounts of CCCP diluted in EasySep but found that increased amounts of CCCP correlated with artificially increased fluorescence reads. From the literature, the team learned that EasySep contains Fetal Bovine Serum (FBS), which autofluoresces and affects the measurements. To prevent this, the CCCP was instead diluted in DMSO, not only to prevent autofluorescence, but also to better permeate the cellular and mitochondrial membranes.

For the positive control, we tried various inhibitors of Complex V, which should lead to an increase in Δψm. We first used oligomycin, but found that it exhibits a dose-dependent autofluorescence. We then tried potassium cyanide, but this decreased the fluorescence. Finally, the team tried Venturicidin A, experimenting with different concentrations and incubation times. Although the results are promising, we are still finalizing the best conditions.

To conclude, we’ve optimized conditions at the cellular and measurement levels, and we are optimistic about the usefulness of this study to predict an increased risk of sepsis.