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Objective

An electron paramagnetic resonance EPR technique using the spin probe cyclic hydroxylamine 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine CMH was introduced as a versatile method for high precision quantification of reactive oxygen species, including the superoxide radical in frozen biological samples such as cell suspensions, blood or biopsies.

Materials and Methods

Loss of measurement precision and accuracy due to variations in sample size and shape were minimized by assembling the sample in a well-defined volume. Measurement was carried out at low temperature 150 K using a nitrogen flow Dewar. The signal intensity was measured from the EPR 1st derivative amplitude, and related to a sample, 3-carboxy-proxyl CP• with known spin concentration.

Results

The absolute spin concentration could be quantified with a precision and accuracy better than ±10 µM k = 1. The spin concentration of samples stored at −80°C could be reproduced after 6 months of storage well within the same error estimate.

Conclusion

The absolute spin concentration in wet biological samples such as biopsies, water solutions and cell cultures could be quantified with higher precision and accuracy than normally achievable using common techniques such as flat cells, tissue cells and various capillary tubes. In addition; biological samples could be collected and stored for future incubation with spin probe, and also further stored up to at least six months before EPR analysis, without loss of signal intensity. This opens for the possibility to store and transport incubated biological samples with known accuracy of the spin concentration over time.



Autor: Kirsti Berg , Madelene Ericsson , Mikael Lindgren, Håkan Gustafsson

Fuente: http://plos.srce.hr/



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