BD Horizon™ Fixable Viability Stain 440 UV, 200 μg

Description

• Recommended Assay Procedures Preparation Bring FVS440UV dye powder and 363 μL of fresh cell culture-grade Dimethyl Sulfoxide (DMSO; eg, Sigma D2650) to room temperature
• Add 363 μL of DMSO and vortex solution well
• Inspect the solution and repeat vortex until the stock dye has fully dissolved
• This is the Stock Solution
• Storage Upon arrival, store the dry dye desiccated and protected from light at -80°C until use
• After reconstitution with DMSO, store the Stock Solution at -20°C in small aliquots
• Do not use reconstituted dye after 90 days of storage
• Please discard the dye solution after 90 days post reconstitution with DMSO
• Cytometry Requirements UV laser-equipped Flow Cytometers (eg, BD LSRFortessa™, BD LSRFortessa™ X-20, or BD™ LSR II) can be used
• This dye can be read out of filters commonly used for DAPI or Hoechst dyes (eg, 450/50)
• This dye is also compatible with filter sets intended for BD Horizon™ BUV395 (eg, 379/28), though more dye may need to be used to achieve the same resolution
• See 'Procedure' for more information
• Fluorescence compensation is best achieved using a sample of the cells of interest stained with FVS440UV
• When designing multicolor panels, please be aware of spillover into the BD Horizon™ BV421 and APC channels
• Panels should be optimized to take this spillover into account
• We recommend titrating the dye and using the lowest possible concentration that provides adequate resolution of live and dead populations for the cell type of interest to reduce spillover
• Procedure Fixable Viability Stain 440UV labeling of cells Prepare cells for flow cytometry staining using sodium azide-free buffers
• Wash cells one time in sodium azide- and protein-free Dulbecco's Phosphate Buffered Saline (1 x DPBS)
• Resuspend cells at 1-10 x 10^6 cells/ml in sodium azide- and protein-free 1 x DPBS
• Add 1 μL of BD Horizon™ Fixable Viability Stain 440UV Stock Solution for each 1 mL of cell suspension (1:1000) and vortex immediately
• Note: We recommend titrating the dye for optimal performance, as different cell types and different applications can result in a wide degree of variability in staining
• Note: If the dye is to be analyzed using a BD Horizon BUV395 filter set (eg, 379/28 bandpass filter), we recommend using at least 2-4 μL per 1 mL of cell suspension as a starting point for titration
• Incubate the mixture for 10-15 minutes at room temperature protected from light
• Optional: Alternatively, incubate mixtures at 37°C for 5-7 minutes or 2-8°C for 30-60 minutes
• Wash cells twice with 2 ml of BD Pharmingen™ Stain Buffer (FBS) (Cat
• No
• 554656) or the equivalent
• Decant the supernatant and gently mix to disrupt the cell pellet
• Resuspend the cells in Stain Buffer (FBS) or equivalent
• Stain, fix and permeabilize cells as desired for downstream applications
• Notes: Each user should determine the optimal concentrations of reagents, cells, and conditions for the assay of interest
• We recommend titrating the reagent in early experiments to obtain optimal results
• The reactivity of the free dye is quenched by washing with buffer containing protein (eg, FBS or BSA)
• Cells may be stained in bulk prior to freezing or staining with fluorescent antibodies
• BD Horizon™ Fixable Viability Stain 440UV can be used in intracellular staining assays that require fixation with formaldehyde and permeabilization with methanol and detergents such as those used for BD Phosflow™ staining (eg, BD Phosflow™ Perm Buffer III, Cat
• No
• 558050), intracellular cytokine staining (eg, BD Cytofix/Cytoperm™ Fixation/Permeablization Kit, Cat
• No
• 554714), or transcription factor staining (eg, BD Pharmingen™ Transcription Factor Buffer Set, Cat
• No
• 562574)
• Apoptotic cells can show variable staining
• We recommend co-staining with, eg, Annexin V FITC (Cat
• No
• 556419) or Alexa Fluor™ 647 Rabbit Anti-Active Caspase-3 (Cat
• No
• 560626) if further analysis is desired for the apoptotic cells.