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

1.Prepare cells for flow cytometry staining using sodium azide-free buffers.

2.Wash cells one time in sodium azide- and protein-free Dulbecco"s Phosphate Buffered Saline (1× DPBS).

3.Resuspend cells at 1-10 × 10^6 cells/ml in sodium azide- and protein-free 1× DPBS.

4.Add 1 µl of BD Horizon™ Fixable Viability Stain 440UV Stock Solution for each 1 ml of cell suspension (1:1000) and vortex immediately.

a.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.

b.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.

5.Incubate the mixture for 10-15 minutes at room temperature protected from light.

a.Optional: Alternatively, incubate mixtures at 37°C for 5-7 minutes or 2-8°C for 30-60 minutes.

6.Wash cells twice with 2 ml of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or the equivalent.

7.Decant the supernatant and gently mix to disrupt the cell pellet.

8.Resuspend the cells in Stain Buffer (FBS) or equivalent.

9.Stain, fix and permeabilize cells as desired for downstream applications.

Notes:

1.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.

2.The reactivity of the free dye is quenched by washing with buffer containing protein (eg, FBS or BSA).

3.Cells may be stained in bulk prior to freezing or staining with fluorescent antibodies.

4.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).

5.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.