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

Protocol summary

1. Prepare samples in HHBS (0.5 mL/assay)
2. Replace with  HHBS 
3. Add Stain It™ Orange 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™ Orange stock solution (500X):
Add 200 µL DMSO (Component B) into the vial of Stain It™ Orange (Component A) to make 500X Stain It™ Orange stock solution.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

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

1. Prepare cells for flow cytometry staining 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™ Orange 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).