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. This dye can be read out of filters commonly used for BD Horizon™ Brilliant Violet 510, BD Horizon™ V500, or AmCyan (e.g., 525/50). Fluorescence compensation is best achieved using a sample of the cells of interest.

Procedure

Fixable Viability Stain 510 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 (1X DPBS).

3.        Resuspend cells at 1x10^6 cells/ml in sodium azide- and protein-free 1X DPBS.

4.        Add 1 μl of the BD Horizon™ Fixable Viability Stain 510 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 15 minutes at room temperature protected from light.

a.        Optional: Incubate the cells and dye mixtures at 2-8°C for 30-60 minutes. Alternatively, incubate mixtures at 37°C for 5-7 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.