Nitric oxide (NO) was measured by the Griess staining
method. At 48 and 72 hours, we collected the supernatant
(cell culture media) of the 4T1 cells that had been exposed
to different doses of the exosomes (0.1, 0.5 and 1 mg/
ml). We deproteinized 400 μl of supernatant by adding 6
mg of zinc sulfphate (Sigma-Aldrich, Canada). The vials
were centrifuged at 4˚C and 12 000 rpm for 12 minutes.
Then, 100 µl of the supernatant of the deproteinized
samples were added to the wells of the 96-well plate and
100 µl of vanadium chloride (Sigma-Aldrich, Canada),
50 µl of sulphanilamide (Sigma-Aldrich, Canada) and 50
µl of N-(1-Naphthyl) ethylenediamine dihydrochloride
(NEDD, Sigma-Aldrich, Canada) were added to each well.
The plate was incubated for 30 minutes at 37˚C. Next,
100 µl standard sodium nitrate solution at concentrations
of 0, 6, 12.5, 25, 50, 100 and 200 µM, was prepared and
we added vanadium chloride, sulphanilamide and NEDD
to the standard wells, which was similar to the approach
used for the experimental samples. The standards and the
samples were read at 540 and 630 nm with an ELISA
reader (Stat Fax 2100, USA) (18 (link)).