Flow cytometric analysis of live and fixed/permeabilized human peripheral blood mononuclear cells, combined and stained with Ghost Dye™ Violet 450 Viability Dye. Viable gate is indicated.
1. Prepare the following reagents with reverse osmosis deionized (RODI) or equivalent grade water:
a. 1X PBS (azide- and protein/serum-free)
b. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
2. Remove Ghost Dye™ from -20°C and bring to room temperature.
3. Collect cells by centrifugation and aspirate supernatant.
4. Wash cells by centrifugation in excess 1X PBS. Repeat if necessary.
5. Resuspend cells to a concentration of 1-10 x 106/mL in 1X PBS.
6. Centrifuge the Ghost Dye™ before opening then add 1 uL for each 1 mL of cell suspension and vortex immediately.
7. Incubate for 30 minutes at 4°C protected from light.
8. Wash by centrifugation in excess incubation buffer. Discard supernatant. Repeat.
9. Cells can then be stained, fixed and/or permeabilized based upon experimental design.
Ghost Dye™ Violet 450 Viability Dye is excited by the violet (405 nm) laser line and has a peak emission of 450 nm that can be detected using 440/40 or 440/50 band pass filters commonly used for detection of violetFluor™ 450, Pacific Blue, etc.
Store at -20°C desiccated and protected from light. This product is stable for 12 months. Aliquot to avoid excessive freeze-thaw cycles.
Ghost Dye™ Violet 450 Viability Dye is used to discriminate viable from non-viable mammalian cells in flow cytometry applications. Ghost Dye™ Violet 450 Viability Dye irreversibly binds free amines available on the cell surface as well as intracellular free amines exposed in cells with compromised cell membranes. Non-viable cells with loss of membrane integrity will react with significantly more Ghost Dye™ Violet 450 Viability Dye than healthy cells in the same sample. Cells that exhibit increased fluorescence intensity can be excluded from analysis.