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 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 12 months. Aliquot to avoid excessive freeze-thaw cycles.
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