Description
Preparation Bring FVS450 dye powder and 400 μl of fresh cell culture-grade Dimethyl Sulfoxide (DMSO; eg. Sigma D2650) to room temperature. Add 400 μl of DMSO and vortex solution well. Inspect the solution and repeat vortex until the stock dye has fully dissolved. Storage Upon arrival, store the dry dye at -80°C until use. After reconstitution with DMSO, store the solution at -20°C. The dye solution can be used for up to four freeze-thaw cycles. Aliquots (eg, ~100 μl aliquots) can be made and stored at -20°C when required for smaller experiments. Do not use reconstituted dye after 40 days of storage. Please discard the dye solution after 40 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. Fluorescence compensation is best achieved using BD™ CompBeads Anti-Mouse Ig, κ/Negative Control (FBS) Compensation Particles Set (Cat. No. 552843) stained with BD Horizon™ V450 Mouse Anti-Human CD3, CD4, or CD19 antibodies. Alternatively, BD™ CompBeads Anti-Rat Ig, κ/Negative Control (FBS) Compensation Particles Set (Cat. No. 552844) stained with BD Horizon™ V450 Rat Anti-Mouse CD3, CD4, or CD19 antibodies can be used. Procedure Fixable Viability Stain 450 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 ~1-10 x 10^6 cells/ml in sodium azide- and protein-free 1X DPBS. 4. Add 1 μl of the Fixable Viability Stain 450 stock solution for each 1 ml of cell suspension and vortex immediately. 5. Incubate the mixture for 10-15 minutes at room temperature protected from light. Optional: Incubate the cells and dye mixtures at 2-8°C for 20-30 minutes (may be more desirable in mouse cell applications). Alternatively, incubate mixtures at 37°C for 5-7 minutes (eg, for BD Phosflow™ applications). 6. Wash cells once or 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: • The reactivity of free dye is quenched by washing with buffer containing protein (eg, FBS or BSA) prior to staining with fluorescent antibodies. • Fixable Viability Stain 450 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, Cat. No. 558050, BD Phosflow™ Perm Buffer III) or intracellular cytokine staining (eg, Cat. No. 554714, BD Cytofix/Cytoperm™ Fixation/Permeablization Kit). • Cells may be stained in bulk prior to freezing or staining with fluorescent antibodies. Each user should determine the optimal concentrations of reagents and cells and conditions for the assay of interest.
1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
3. BD Horizon V450 has a maximum absorption of 406 nm and maximum emission of 450 nm. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
4. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
5. Cy is a trademark of GE Healthcare.
6. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
