Phosphate Buffered Saline (PBS), 10X

1. Reconstitute the vial of CFSE with 200 µL DMSO to create a 2500X stock concentrate at 0.25 mg/mL. Mix by swirling or tilting the vial, allowing the DMSO to travel around the base of the amber vial until completely dissolved. At room temperature, the reagent should be dissolved within a few minutes. 2. If storing the stock concentrate for future use, prepare small aliquots (20 µL) to avoid freeze-thaw cycles. The stock concentrate will be stable for 6 months when protected from light and stored at or below -20°C. 3. Create the 10X working solution by diluting the 2500X stock solution 1:250 in sterile PBS; e.g., add 4 µL stock to 996 µL PBS. Store the working solution on ice up to 2 hours protected from light. Do not use media to dilute the CFSE as it will quench the fluorescent signal., 4. Prepare cells at 1-2 x 107 in 1.8 mL sterile PBS., 5. Create 1 control tube of unstained cells at 1-2 x 107 in 2 mL sterile PBS. These cells will be used to compensate the flow cytometer to ensure that stained cells shift along the FL1 axis., 6. Stain cells at a final concentration of 0.1 µg/mL (0.18 µM) of CFSE in the cell culture. Add the 10X working solution to the cells at a dilution of 1:10. For example, add 200 µL 10X CFSE working solution into 1.8 mL cell suspension. Mix by inverting or vortexing the vial. The optimal concentration of CVSE may vary among cell types. The concentration of CFSE and the incubation time should be adjusted for cell line to adequately stain them. Excessive staining may cause problems when compensating the instrument. Do not add CFSE to the control tube., 7. Incubate 15 minutes at room temperature., 8. Add 1 mL cell culture media to stop the reaction., 9. Incubate 5 minutes., 10. Wash the cells once or twice by centrifugation and discard the supernatant., 11. Resuspend in cell culture media such that each tube contains the desired level of target cells, or resuspend in PBS and fix cells with formaldehyde., 12. Analyze cells, or incubate at 37°C up to 1 hour until ready for additional staining or further experimentation., 13. Analyze with a flow cytometer equipped with a 15 mW 488 nm argon laser: excitation at 492 nm; emission at 520-540 nm in FL1. Stained cells appear green.