Cell culture media that contain high levels (more than 30 µg/litre) of biotin (e.g., RPMI, which contains 200 µg/litre biotin) will compete with biotinylated reporter antibodies for the streptavidin–PE reporter molecules. This will result in lower mean fluorescence intensity (MFI) readings and reduced sensitivity. For such media, a wash step must be incorporated into the assay procedure. Supernatants from cells grown in media containing lower amounts of biotin, such as DMEM (no biotin), AIM V (3.5 µg/litre), IMDM (13 µg/litre), and 10% FBS (3.4 µg/litre) can be processed using the Standard Assay Protocol.