Development of a Methodology to Determine the Effect of Interleukin-2 on the Mitochondrial Membrane Potential of Human Fibroblasts as Measured by Confocal Microscopy
Creators: name: Pettengill, Marilyn J.
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 1999

description: There are known mitochondrial diseases in which ATP production is reduced, causing fatigue and inability of cells to respire normally. There are also known inhibitors and drugs that can interfere with the potential of mitochondria to produce ATP. When some of these inhibitors are applied to cells whose mitochondrial membrane potential is being measured by confocal microscopy, a change in the fluorescence, indicative of a change in mitochondrial potential can be viewed and quantified. The use of cytokines, such as Interleukin-2 in cancer therapy and other immunotherapy is known to cause fatigue, over and above the disease being treated. Interleukin-2 involvement has also been implicated in some chronic illness, of which fatigue plays a major role. By adding Interleukin-2 to living fibroblasts in culture, one can determine if this cytokine has an impact on mitochondrial potential and thus a reduction or change in ATP production. Normal human fibroblasts were cultured and their average cytoplasmic and mitochondrial membrane potentials and percent areas were determined using confocal microscopy with tetramethylrhodamine methyl ester (TMRM) as a fluorescent probe. TMRM is a cationic dye specific for mitochondria that distributes according to the Nernst equation. Using software created at the University of North Carolina, Chapel Hill, membrane potentials and areas were calculated. These means were accumulated in time increments of 15 minutes up to 2 hours and at 24 hours. Controls for the confocal microscopy process of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and oligomycin were used. Interleukin-2 in concentrations of 10 ng/ml, 100 ng/ml and 200 ng/ml were added and measured in the same time intervals, as described for the untreated fibroblasts. CCCP was also added to Interleukin-2 100 ng/ml samples. Ideally, following one cell over time should give the most accurate results, however, fading of the fluorescent probe after taking multiple images of the same cell is not uncommon. The variability in normal fibroblasts was great enough that determination of change from the addition of Interleukin-2 was difficult. Three analysis methods were created to overcome these limitations. Methodone accumulated data from images of untreated and IL-2 treated new cells. These were grouped in the time categories listed previously. The untreated and treated means were calculated and compared using Student's T-Test. Method two accumulated data from images of untreated and treated new cells followed by one subsequent image of the same cell. These also were accumulated by mean within time category and displayed using bar charts. The final option was to follow a single cell over time. An untreated cell was matched with a treated cell of like cellular characteristics and the changeover time compared. The results in all three methods indicate a change in the mitochondrial membrane potential that may be attributed to the presence of Interleukin-2. In addition, the normal decrease in mitochondrial potential due to CCCP does not occur as rapidly in the presence of Interleukin-2.