Ghost Dye™ viability dye (see http://www.tonbobio.com/ghost­dyes for available formats)

Other Materials Required

  • 1X PBS (azide­free, protein/serum­free)
  • Flow Cytometry Staining Buffer (Stain Buffer) (1X PBS with 2% FBS, 0.09% Na-Azide)
  1. Remove Ghost Dye vial from freezer and allow to equilibrate to room temperature.
  2. Quick spin Ghost Dye vial before opening.
  3. Prepare azide­ and protein/serum-­free 1X PBS for labeling procedure.
  4. Wash cells twice in 1­-2 mL azide­ and protein/serum­-free 1X PBS. Spin at 300­-400 x g for 5 minutes at room temperature and decant supernatant.
  5. Resuspend cells to a concentration of 1-­10 x 10^6 /mL in azide­ and protein/serum-free 1X PBS.
  6. Add 1 μL of Ghost Dye solution for each 1 mL of cell suspension and vortex immediately. Note: Ghost Dyes are formulated in DMSO, pipet carefully and slowly.
  7. Incubate for 30 minutes at 2­-8°C protected from light.
  8. Wash cells 1­-2 times with 1-­2 mL Stain Buffer. Washing with a protein containing buffer allows removal of any unreacted dye prior to staining with fluorescent antibodies.
  9. Cells can be subsequently stained, fixed and permeabilized according to user protocol.

Note: Cells labeled with Ghost Dyes can be cryopreserved for later use or used in intracellular staining protocols without any loss of fluorescence intensity.

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