Cell Explorer™ Live Cell Labeling Kit *Green Fluorescence with 405 nm Excitation*

Protocol summary

1. Prepare cells in growth medium
2. Remove growth medium
3. Add CytoCalcein™ Violet 500 working solution (100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate)
4. Incubate the cells at 37°C for 30 minutes to 1 hour
5. Wash the cells
6. Examine the specimen under fluorescence microscope with Violet filter set or flow cytometer with 525/40 nm filter (AmCyan channel)

Important notes
Thaw all the components at room temperature before opening.

1. CytoCalcein™ Violet 500 stock solution:
Add 20 µL of DMSO into the vial of CytoCalcein™ Violet 500 (Component A) and mix well to make CytoCalcein™ Violet 500 stock solution. Note: 20 µL of CytoCalcein™ Violet 500 stock solution is enough for 1 plate. Note: Unused CytoCalcein™ Violet 500 stock solution can be aliquoted and stored at < -20 ^oC for more than one month if the tubes are sealed tightly. Avoid repeated freeze-thaw cycles and protect it from light.

Add 20 µL of CytoCalcein™ Violet 500 stock solution into 10 mL of HHBS (Component B) and mix well to make CytoCalcein™ Violet 500 working solution.

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

1. Remove the growth medium.
   
   
2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) CytoCalcein™ Violet 500 working solution into the cell plate.
   
   
3. Incubate the cells in a 37°C, 5% CO₂ incubator for 30 minutes to 1 hour.
   
   
4. Remove the CytoCalcein™ Violet 500 working solution from the cells, wash the cells with HHBS (Component B) for 2 to 3 times, and replace with HHBS.
   
   
5. Analyze the cells using a fluorescence microscope  with Violet filter set or flow cytometer with 525/40 nm filter (AmCyan channel). Note: Alternatively, cells can be fixed at this point for later imaging (fluorescence intensity might be decreased upon fixation).