Microenvironment contamination occurs from all lipophylic dyes tested. HL60 cells were stained in PBS without serum with CFSE (0.5μM),³¹  DiI, DiR, or PKH26 (2μM) for 10 minutes and washed twice in PBS, 2% FCS. Stained cells were either directly plated on a confluent layer of fibroblasts (GP293T; condition 0 hours) or incubated for 24 hours in standard culture conditions before being plated on fibroblasts (condition 24 hours). After 24 hours of coculture at 37°C, cells were recovered by trypsinization, stained for hCD45 + hCD33 (both PE in the case of DiR, APC for all other dyes), and analyzed by flow cytometry. The absolute amount of fluorescence associated with the fibroblasts is displayed as a percentage of the total fluorescence for each dye (average and standard deviation obtained from 4 wells; data are representative of 2 independent experiments). Comparing 0 hours versus 24 hours for each dye, the differences are statistically significant (*P < .004 for all dyes except CFSE where P = .7). After 24-hour coculture, all dye transfers using lipophilic dyes are higher than with CFSE (**P < .001).