Description
BD Horizon™ Fixable Viability Stain 510 (FVS510) is useful for discrimination of viable from non-viable mammalian cells in multicolor flow cytometric applications. This dye reacts with and covalently binds to cell-surface and intracellular amines. Permeable plasma cell membranes, such as those present in necrotic cells, allow for the intracellular diffusion of the dye and covalent binding to higher overall concentrations of amines than in non-permeable live cells. Therefore, necrotic cells present in a typical in vitro assay label with higher levels of dye increasing their fluorescence intensity 10-20 fold over that of viable cells. The labeled cells can be fixed with formaldehyde for downstream decontamination, freezing and/or permeabilization and subsequent intracellular staining while maintaining stable viability stain fluorescence.
BD Horizon™ Fixable Viability Stain 510 is excited by the Violet laser (with an excitation maximum of 408 nm) and has a fluorescence emission maximum of 512 nm.
Suggested Companion Products
Transcription Factor Buffer Set RUO 25TestsCat No: 562725
BD Cytofix/Cytoperm™ Fixation/Permeablization Kit RUO 250TestsCat No: 554714
APC Annexin V RUO 200TestsCat No: 550475
Fixation Buffer RUO 100mLCat No: 554655
Perm/Wash Buffer I RUO 125mLCat No: 557885
Perm Buffer III RUO 125mLCat No: 558050
Stain Buffer (FBS) RUO 500mLCat No: 554656
Transcription Factor Buffer Set RUO 100TestsCat No: 562574
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Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
Preparation
Bring FVS510 dye powder and 260 μl of fresh cell culture-grade Dimethyl Sulfoxide (DMSO; e.g. Sigma D2650) to room temperature. Add 260 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
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.
Notes:
1.Each user should determine the optimal concentations 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 (e.g., FBS or BSA).
3.Cells may be stained in bulk prior to freezing or staining with fluorescent antibodies.
4.BD Horizon™ Fixable Viability Stain 510 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 (e.g., Cat. No. 554714, BD Cytofix/Cytoperm™ Fixation/Permeablization Kit), or transcription factor staining (e.g., 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) if further analysis is desired for the apoptotic cells.
