BODIPY™ 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene)

BODIPY 493/503 NHS Ester (Succinimidyl Ester) (Cat. No. D2191) is the amine‐reactive derivative of BODIPY 493/503 (4,4‐Difluoro‐1,3,5,7,8‐Pentamethyl‐4‐Bora‐3a,4a‐Diaza‐s‐Indacene) (Cat. No. D3922). The reactive group allows for D2191 to be used as a labeling reagent to label proteins or other molecules to covalently attach the dye to accessible primary amines. If using this as a live cell reagent, the reactive group may allow for better retention as it will covalently attach to any primary amines on any cellular material it may come into contact with.

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We do not have a protocol for using BODIPY 493/503 (D3922) on paraffin section. This product is typically used on live cells.

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Invitrogen BODIPY 493/503 dye (Cat no. D3922) being a hydrophobic dye does not disperse readily into aqueous solutions. To consistently stain cells with this dye, follow this protocol:

- Place your cells in a half-volume of buffer or media. For this example, if you have 1x10E6 cells/mL in 1 mL, place them as 1x10E6 cells into a 0.5 mL volume (2x10E6 cells/mL).
- Make a 2X solution of Invitrogen BODIPY 493/503 dye in 0.5 mL volume of the same pre-warmed buffer or media (no cells, no BSA or serum) and mix vigorously to mechanically emulsify this solution.
- Either vortex, sonicate or shake vigorously by hand and then IMMEDIATELY add this to the solution of cells and then mix or roll gently.
- For adherent cells, swirl gently or pipette up and down.
- When the 0.5 mL of the 2X dye/buffer solution is added to the 0.5 mL of cells/buffer, the final dye concentration will be 1X and the cells will be at their appropriate final density of 1x10E6 cells/mL.

Final dye concentrations can range from 1 to 10 µM with incubation times ranging from a few mins up to 30 mins; the optimal final concentration and incubation times should be optimized per the application.

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For Invitrogen BODIPY 493/503 dye (Cat. No. D3922), one may make a stock solution of 3.8 mM (1 mg/mL) in high-quality anhydrous DMSO. After resolubilizing, store the stock solution frozen, desiccated, and protected from the light. Solutions up to 1mM can also be made in absolute ethanol.

Please note that both the DMSO and ethanol should be of high quality and anhydrous or the compound may not dissolve.

For long-term storage, dissolve the dye at 1 mM using absolute ethanol, make smaller aliquots in separate vials, and then evaporate off the ethanol using a vacuum pump. Store the solid dye frozen and protected from the light.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.