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 fixed, permeabilized, and immunostained based upon experimental design and recommended protocols.
10. Exclude cells with high Ghost Dye™ fluorescence from analysis. These were non-viable cells at the time of fixation. See details below for excitation and emission specifications.
Ghost Dye™ Violet 510 Fixable Viability Dye is excited by the violet (405 nm) laser line and has a peak emission of 510 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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