Cell Explorer™ Live Cell Labeling Kit *Red Fluorescence*

1. Prepare cells in growth medium
2. Remove the medium

3. Add Calcein Deep Red™ working solution (100 µL/well for 96-well plates or 25 µL/well for 384-well plates)

4. Incubate cells at 37°C for 30 minutes to 2 hours
5. Wash the cells
6. Examine the specimen under under fluorescence microscope with Cy5 filter (Ex/Em = 646/660 nm)

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

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

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Add 20 µL of DMSO into the vial of Calcein Deep Red™ (Component A) and mix well to make Calcein Deep Red™ stock solution. Note: 20 µL of Calcein Deep Red™ stock solution is enough for 1 plate. Note: Unused Calcein Deep Red™ stock solution can be aliquoted and stored at < -20 ^°C for 2 weeks if the tubes are sealed tightly. Avoid repeated freeze-thaw cycles and protect from light.

Add 20 µL of Calcein Deep Red™ stock solution into 10 mL of HHBS (Component B) and mix well to make Calcein Deep Red™ working solution. Protect from light.

1. Remove the growth medium from the cell plates. Note: It is important to remove the growth medium in order to minimize the background fluorescence and increase the signal to background ratio.

2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) Calcein Deep Red™ working solution into the cell plate.

3. Incubate the cells in a 37°C, 5% CO₂ incubator for 30 minutes to 2 hours.

4. Remove the Calcein Deep Red™ working solution from the cells.

5. Wash the cells with HHBS (Component B) for 2 to 3 times, and replace with HHBS.
6. Image the cells using a fluorescence microscope with Cy5 filter (Ex/Em = 646/660 nm).