Something in your buffer have oxidizing activity of reduced form cyt.c. The assay of cyt. c is measuring change in absorbance at 550nm. It is reduced form of cyt.c. When I measured cyt.c oxiase activity there were no assay kits because it was more than 50 years ago. I learned the principal of assay methods in "Methods in Enzymoloy, vol. X". At first, I had to reduce cyt.c solution using some reducer (I forgot the chemical), then pass a column packed Dowex resin to remove reducing agents.

The principle of the assay method is measuring the decrease of absorbance at 550nm (it is absorbance of reduced form of cyt. c) by the enzyme activity of cyt. c oxidase in your sample lysate. If your blank showed oxidizing activity, it meane something which have oxidizing activity of reduced cyt.c (maybe bacteria or fungi, I suppose) was present.

Than you very much for your insight. Frankly, the only thing that could be contaminated is the diluition buffer. Could I simply replace it with distilled water?

No! To measuring enzyme activity, pH is important, so you have to use buffer. My sayings mean your PBS with Triton X-100 might be contaminated with bacteria of something. So that you should make new PBS with Triton X-100. I thought your dilution buffer means the PBS with Triton X-100. If not, what are you using in your measurement for blank assay?

Thank you again for your insight.

I'm using a pre-prepared assay kit (bought from abcam). Unfortunately I couldn't find any info on the components of the diluition buffer.

To be as clear as possible, I'll try to recap the contents of the wells:

- blank: 120 uL of the reduced form of cyt c and 10 uL of diluition buffer

-samples: 120 uL of the reduced form of cyt c and 10-15 uL of lisates prepared with PBS with Triton X-100 (the exact volume changes depending on the protein concentration in the sample)

The "10-15 uL of lisates prepared with PBS with Triton X-100" is suspicious. Is the PBS with Triton X-100 freshly made? If you used old, stocked PBS, you had better to make new PBS.