IMPROVED SAMPLE HANDLING FOR CELL STUDIES BY HIGH FREQUENCY EPR

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Nitroxide reduction experiments are usually carried out at X-band (8.8-9.5 GHz) with a relatively large number of cells (typically 107 cells in 100 l). Such a large number of cells is not always available for experimentation. For example, it is of interest to probe the cellular metabolism during their transformation by adenovirus when, initially, only few cells are involved. Additionally, high concentrations of cells in X-band experiments often result in inhomogeneous cell distribution in a sample tube and eventually in long diffusion paths for the metabolites and complicated apparent kinetics. We already showed (Rocky Mountain Conference, 1994) that the number of cells in the nitroxide reduction experiments can be reduced to only 900 by use of a W-band (95 GHz) EPR spectrometer developed at the Illinois EPR Research Center (IERC). By applying a resonator with a higher Q-value, careful sample positioning, and lineshape fitting software we were able to decrease this number to six cell s in a sample. We have measured reduction rate of TEMPONE (T=36.6 oC) at both X-band (3x106 cells in 30 l) and W-band (6 cells in 7.5 nanoliters). The rate measured from W-band experiments (3.5x10-16 molesxmin-1xcell-1) was essentially the same as that found from X-band (3.3X10-16 molesxmin-1xcell-1). From signal-to-noise considerations, we infer that it will be possible to observe nitroxide reduction by a single cell at W-band. This would open the path towards measurements of cell redox changes in which only a few cells participate. This is an ongoing study.