16.Assay for superoxide anion(O2-) release(Ingvar Ferby)
Material and equipment:
i) Cytochrome C (cyt.C), type VI (oxidized form)
ii) Superoxide dismutase (SOD)(Stock: 5000U/ml in 0.15M NaCl at -20degree)
iii)Siliconized microtubes.
iiii) Spectrophotometer able to detect absorbance at dual wavelenghts(550nm and 540nm)
Preparation and challengeing of cells:
1) Cells are prepared and suspended in a colourless medium (ex:10mM Hepes pH 7.4 buffered HBSS) to an approximate concentration of 5x106
cells per ml (for leukocytes).
2) Aliquots of 0.5 ml cellsuspention are preincubated for >10 min at 37ⅳ and challenged with 50 μl ligand solution containing freshly dissolved cytochrome C enough to yield a final concentration of 1 mg cyt.C per ml. Cytochrome C can not be stored in solution but must be freshly dissolved for every assay. Blanks are made by including 45 U of superoxide dismutase(SOD) just prior to stimulation, otherwise treating them as the samples. The oxygen radicals released by the cells will reduce cyt. C. SOD inhibits this reduction.
3) The release of superoxide anion is quick and in most cases completed after 3 min. Terminate the stimulation by quick centrifugation (20 sek.) on table type microfuge, and transfer of supernatant to new tubes.
Measure of released O2-:
4) The reduction of cyt.C is mesuared as an increase in absorbance at 550 nm.
The most correct procedure is to measure the difference in absorbance between 550 nm and 540 nm(Abs550- Abs540) in the recovered poststimulatory supernatants.
5) The released amount of O2- . is calculated using Lambert-Beers law. [A = ｶ x c x d], where A is (Abs550- Abs540), c the concentration in M, d the light path of the cuvette in cm and ｶ the extriction coefficient. ｶ in this case is 19.1 x 103 M-1cm-1(Meth.in Enz., vol. 132 p 407). One mole of cyt.C reduces one mole of O2- .
Warnings:
a) Perform the absorbance measurements on the poststimulatory supernatants as soon as possible, since in my experience, the reduced cyt.C tend to decline fairly with time.
b) O2- release by leukocytes is affected significantly by fenomena such as adherance and aggregation. I therefore recommend the use of siliconized tubes when incubating the cells, in order to minimize this effect.