Cell Explorer™ Live Cell Tracking Kit *Deep Red Fluorescence*

Protocol summary

1. Prepare samples
2. Add 10X Track It™ Deep Red working solution (10 µL/well)
3. Stain the cells at 37 ^oC for 15 minutes to 1 hour
4. Wash the cells
5. Examine the specimen under fluorescence microscope with Cy5 filter or flow cytometer with 660/20 nm filter (APC channel)

Important notes
Thaw all the components to room temperature. Centrifuge the component A briefly before opening.

Dilute 500X Track It™ Deep Red DMSO stock solution (Component A) into Assay Buffer (Component B) to make a 10 to 25X Track It™ Deep Red working solution. The working solution should be prepared enough for all the wells at 10 µL/well with the appropriate concentration. For example, to get a 10X final concentration of Track It™ Deep Red for one 96-well microplate, dilute 20 µL of 500X Track It™ Deep Red DMSO stock solution into 1 mL of Assay Buffer (Component B) to make 1 mL of 10X Track It™ Deep Red working solution. Note: The final concentration of the Track It™ Deep Red working solution should be empirically determined for different cell types and/or experimental conditions. It is recommended to test at the concentrations at least over a ten fold range. Note: The unused portion of the Track It™ Deep Red stock solution should be stored at -20 ^oC. Avoid repeated freeze/thaw cycles.

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

1. Add 10X Track It™ Deep Red working solution to the cell wells which should be equal to 1/10 of the volume of cell culture medium. For example, for a 96-well plate, add 10 µL/well of 10X Track It™ Deep Red working solution into the cells.
   
   
2. Incubate the cells in a 37°C, 5% CO₂ incubator for 15 minutes to 1 hour.
   
   
3. Wash cells with Hanks and 20 mM Hepes buffer (HHBS) or an appropriate buffer.
   
   
4. Fill the cell wells with growth medium.
   
   
5. Analyze the cells using a fluorescence microscope with Cy5 filter or flow cytometer with 660/20 nm filter (APC channel).