Flow cytometric analysis of live and fixed/permeabilized peripheral blood mononuclear cells, combined and stained with Ghost Dye™ Violet 540 Viability Dye. Viable gate is indicated.Learn more about how we get our images
Ghost Dye™ Violet 540 Viability Dye is used to discriminate viable from non-viable mammalian cells in flow cytometry applications. Ghost Dye™ Violet 540 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™ 540 Violet Viability Dye than healthy cells in the same sample. Cells that exhibit increased fluorescence intensity can be excluded from analysis.
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 temparature.
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 540 Viability Dye excited by the violet (405 nm) laser line and has a peak emission of 540 nm that can be detected using a 525/50 band pass filter commonly used for detection of AmCyan.
Store at -20°C desiccated and protected from light. This product is stable for 6 months. Aliquot to avoid excessive freeze-thaw cycles.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Ghost Dye is a registered trademark of Tonbo Biosciences.
Discover what’s going on at CST, receive our latest application notes and tips, read our science features, and learn about our products.