Sample Diluent - Non-Mammalian-based

1. AO may be used neat or diluted in diH2O, PBS, or media. ICT’s AO is supplied at 0.5 mL liquid at 1 mM (266 µg/mL). 2. Add AO to the cell sample media at 0.5-5 µM, equal to a final dilution of 1:2,000 – 1:200 in the cells (.05-.5% v/v). For example, if using AO at 1.0 µM in the final cell suspension, it must be diluted 1:1,000. First dilute it 1:100 in PBS or diH2O; e.g., put 10 µL AO into 990 µL PBS or diH2O. Pipette the diluted AO into the cell suspension at approximately 1:10; e.g., put 50 µL diluted AO into 450 µL cell suspension., 3. Incubate 15-30 minutes at 37°C., 4. Wash cells if reagent is too bright., 5. Analyze with fluorescence. Lysosomes will appear yellowish green by illuminating cells with a blue light (488 nm) excitation filter and a green light (540-550 nm) emission/barrier filter. Alternatively, lysosomes will appear red when using an excitation filter of 550 nm (540-560 nm) and a long pass >610 nm emission/barrier filter.

A complex diluent to control background signal in biological samples. Sample Diluent - Non-Mammalian-based is formulated to provide a complex yet protein-friendly environment for the dilution of biological samples (e.g. serum, cell culture media) into the useful range of antibody-sandwich or antigen-down ELISA-format assays. Due to the finite binding capacity of plate-coated proteins (e.g., antibodies, antigens), highly concentrated samples must be diluted in order to obtain absorbance readings within the sensitivity detection limits of the instrument and to create a functional standard curve. Sample Diluent - Non-Mammalian-based minimizes backgrounds and increases assay specificity. It has proven to be highly effective for routine dilution of mouse, porcine, bovine, or rabbit serum samples in antigen-down ELISAs. When testing serum and plasma samples, non-specific adsorption of sample IgG to the ELISA plate surface is a common cause of high background noise. In particular, the glycosylation pattern of porcine serum IgG tends to make porcine samples more ‘sticky’ than IgGs from other species, such as rabbit or mouse. Sample Diluent - Non-Mammalian-based is formulated to reduce this non-specific interaction so that porcine serum samples can be tested without needing a dilution factor beyond 1:100. Sample Diluent - Non-Mammalian-based provides a non-mammalian protein-buffered, neutral pH environment that is highly compatible with antibody-antigen interactions. Antimicrobial agents allow for room temperature bench-top use and extensive storage stability at 2-8°C.