BD Pharmingen™ DAF-FM DA, 500 μg

Description

• Recommended Assay Procedures Preparation Bring DAF-FM DA dye powder and anhydrous Dimethyl Sulfoxide (DMSO) to room temperature
• Add 10 μLof DMSO to dye powder and vortex solution well
• Inspect the solution and repeat vortex until the stock dye has fully dissolved
• This yields a 10 mM stock solution
• Storage Upon arrival, store the dry dye with desiccant and protected from light at -20°C until use
• We recommend a fresh vial of dye be used for each experiment and that reconstituted dye be discarded after use
• However, if stock solutions in DMSO are to be kept for use, they should be stored with desiccant and protected from light at -20°C
• Working solution that have been diluted into aqueous solution from DMSO should not be kept for further use
• Cytometry Requirements Blue laser-equipped flow cytometers (eg, BD FACSCanto™ II, BD LSRFortessa™, BD LSR™ II, or BD Accuri™ C6) can be used. This dye can be read out of filters commonly used for FITC (eg, 530/30-nm bandpass filter)
• Fluorescence compensation is best achieved using stained and unstained samples of the target cells
• When designing multicolor panels, please be aware of spillover into the PE and BD Horizon™ PE-CF594 channels on the blue laser
• If available, collecting fluorescent signals from these fluorochromes using the yellow-green (eg, 561 nm) laser may be advantageous to avoid spillover from DAF-FM fluorescence
• Staining panels should be optimized to take this spillover into account
• Additionally, for multicolor panels, we recommend using the lowest concentration of DAF-FM DA that still provides adequate resolution for the cell type and conditions of interest
• Procedure Staining of Live Cells for Analysis by Flow Cytometry Count cells to determine cell density
• If necessary, adjust cell density to 1 × 10e6 cells/mL in fresh, pre-warmed 1 x DPBS
• Buffers containing serum, BSA, or phenol red may affect the fluorescence of DAF-FM DA
• Therefore, these buffers should be used with caution
• Esterase activity in serum can cleave AM moieties from the dye prior to entry into cells
• Therefore, if serum must be used, it should be heat-inactivated
• Add dye stock solution for a final staining concentration of 1-10 μm and vortex immediately
• We recommend titrating the dye for optimal performance, as different cell types, incubation periods, or culture conditions can result in variability in staining
• Incubate 15-60 minutes at 37°C
• Wash twice with buffer of choice and resuspend cells in analysis or treatment buffer of choice
• For some cell types with low esterase activity, it may be advantageous to incubate cells for another 30-60 minutes to allow complete de-esterification of AM moieties
• In this case, cells should be incubated in a physiologic buffer of choice or complete medium, washed once more, and then resuspended in analysis or treatment buffer of choice
• Treat cells at 37°C for a desired period of time to generate nitric oxide
• A positive control can be generated by treating cells with 1 mM DEA NONOate (Sigma Cat
• No
• D5431) for 30-60 minutes at 37°C
• Wash cells once to remove treatment compounds
• Proceed to analysis by flow cytometry.