CFDA-SE labeling

Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) fluorescent staining is used for long-term cell tracking and quantitation of proliferation, both in vivo and in vitro. Cells were stained with CFDA-SE as previously described.²¹ Briefly, cell pellets were washed three times with PBS, and 1 × 10⁷ cells were incubated in 1 ml of 1 μM CFDA-SE labeling solution for 8 min at 37 °C. After incubation, the labeling reaction was stopped by adding an equal volume of FCS for 1 min. The cells were washed twice with PBS and centrifuged at 1000 r.p.m. for 5 min at room temperature. The cells were then resuspended and incubated in complete RPMI medium for 96 h. The labeled cells were also stained with the nuclear dye, Hoechst, and attached to slides by cytospin (as described above). The percentage of fluorescent positive large L428 cells compared with small cells was quantified by counting 500 cells in three different experiments.