Quantification of Iron (III) in Macrophages

After 24-h exposure to JWH-133 and AM630, cell culture supernatants were collected to measure iron (III). The assay was performed by using the Iron Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Briefly, standards and macrophages supernatant were pipetted into the wells and were incubated with an acidic buffer to allow iron release. Then, an iron probe at 25 °C for 60 min was added, protected from light. Released iron reacted with the chromogen resulting in a colorimetric (593 nm) product, proportional to the iron amount. The optical density was measured at a wavelength of 593 nm by using the DAS Italy plate reader (DAS Italy, Palombara Sabina—Italia.). Iron (II) and Total Iron (II + III) contents of the test samples (nmol/μL) were determined against a standard concentration curve. Iron (III) content can be calculated as: Iron (III) = Total Iron (II + III) − Iron (II).