Cell Explorer™ Live Cell Tracking Kit *Orange Fluorescence*

Protocol summary

1. Prepare samples and remove cells from incubator
2. Add 10 µL/well of 10X Track It™ Orange working solution in each well
3. Stain the cells at 37°C for 15 minutes to 1 hour
4. Wash the cells
5. Examine the specimen under fluorescence microscope with Cy3/TRITC filter or flow cytometer with 575/26 nm filter (PE channel)

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

Dilute 500X Track It™ Orange DMSO stock solution (Component A) into Assay Buffer (Component B) to make a 10X to 25X Track It™ Orange 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 1X final concentration of Track It™ Orange for one 96-well microplate, dilute 20 µL of the Track It™ Orange DMSO stock solution into 1 mL of Assay Buffer (Component B) to make 1 mL of 10X Track It™ Orange working solution. Note: The final concentration of the Track It™ Orange working solution should be empirically determined for different cell types and/or experimental conditions. It is recommended to test at the concentrations that are at least over a ten-fold range.

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

1. Add 10X Track It™ Orange working solution to the cells, the volume 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™ Orange 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 (HBSS) or an appropriate buffer.
   
   
4. Fill the cell wells with growth medium.
   
   
5. Analyze the cells using a fluorescence microscope with Cy3/TRITC filter sets of flow cytometer with 575/26 nm filter (PE channel).