Preparation

Bring FVS575V dye powder and 340 μL of fresh cell culture-grade Dimethyl Sulfoxide (DMSO; eg, Sigma D2650) to room temperature. Add 340 μL of DMSO; 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

Violet laser-equipped Flow Cytometers (eg, BD FACSCanto™ II, BD LSRFortessa™ or BD™ LSR II) can be used. Please note that FVS575V is also excited by the UV laser. This dye can be read out of filters commonly used for BD Horizon Brilliant Violet 605 (eg, 610/20) or Pacific Orange (eg, 575/20). Fluorescence compensation is best achieved using a sample of the cells of interest. When designing multicolor staining reagent panels, please be aware of spillover into the BD Horizon BV510 channel. 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 575V 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 575V 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.

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 575V 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 (e.g., Cat. No. 558050, BD Phosflow Perm Buffer III), intracellular cytokine staining (eg, Cat. No. 554714, BD Cytofix/Cytoperm™ Fixation/Permeablization Kit), or transcription factor staining (eg, Cat. No. 562574, BD Pharmingen Transcription Factor Buffer Set).

5.Apoptotic cells can show variable staining. We recommend co-staining with, e.g., Annexin V APC (Cat. No. 550475) or Alexa Fluor® 647 Rabbit Anti-Active Caspase-3 (Cat. No. 560626) if further analysis is desired for the apoptotic cells.