Live or Dead™ Fixable Dead Cell Staining Kit *NIR Fluorescence*

Protocol summary

1. Prepare samples in HHBS (0.5 mL/assay)
2. Replace with HHBS
3. Add Stain It™ NIR fluorescence to the cell suspension
4. Stain the cells at room temperature or 37°C for 20 - 60 minutes
5. Wash the cells
6. Fix the cells (optional)
7. Examine the sample with flow cytometer and/or fluorescence microscope using the appropriate Excitation/Emission filter 

Important notes
Thaw all the components at room temperature before starting the experiment.

1. Stain It™ NIR fluorescence stock solution (500X):
Add 200 µL DMSO (Component B) into the vial of Stain It™ NIR fluorescence (Component A) to make 500X Stain It™ NIR fluorescence stock solution.

Table 1. Fluorescence spectra properties and suggested excitation laser for flow cytometry analysis

1. Prepare cells using 1X Hanks and 20 mM Hepes buffer (HHBS) or sodium azide-free and serum/protein-free buffer of your choice.
   
   
2. Wash cells once with HHBS or the azide- and serum/protein-free buffer of your choice.
   
   
3. Resuspend cells at 5 - 10 × 10⁶/mL in HHBS or in the azide- and serum/protein-free buffer of your choice.
   
   
4. Add 1 µL of 500X Stain It™ NIR fluorescence stock solution to 0.5 mL of cells/assay and mix it well.
   
   
5. Incubate at room temperature or 37°C, 5% CO₂ incubator for 20 - 60 minutes, protected from light. Note: The optimal stain concentrations and incubation time should be experimentally determined for different cell lines.
   
   
6. Wash cells twice and resuspend cells with HHBS or the buffer of your choice.
   
   
7. Fix cells as desired (optional).
   
   
8. Analyze cells with flow cytometer and/or fluorescence microscope using the appropriate Excitation/Emission filter (see Table 1).