Kinetics and flow cytometric detection of [Cy5-D-R-E-I-M-R]₂-Rh110 peptide uptake (Cy5 fluorescence) and peptide cleavage (Rh110 fluorescence) in U937 cells.

<p>(A) U937 cells were incubated with 10 μM peptide in complete RPMI-1640 medium for increasing time periods (1–240 min) followed by flow cytometric analysis according to the standard protocol as described in Materials & Methods. (B) Visualization of peptide uptake by Cy5 fluorescence microscopy. Equal amounts of peptide-treated (10μM, 90 min) and untreated cells were mixed and subjected to fluorescence microscopic analysis. Cy5 fluorescence is shown in pink, nuclei are counterstained with DAPI (blue). (C) Time kinetics of peptide uptake was calculated as the percentage of peptide (Cy5) positive cells (triangular dots) and the mean fluorescence per cell (round dots). (D) Time kinetics of Separase-related peptide cleavage was calculated as the percentage of Rh110 positive cells (triangular dots) and the mean fluorescence per cell (round dots). (E) Peptide dose (0–20 μM) dependency of Separase-related peptide cleavage. In all experiments, peptide alone (Ac-D-R-E-I-M-R) without dye (Cy5, Rh110) conjugation was used as non-fluorogenic control substrate and cellular autofluorescence gating control. Abbreviations: RFU, relative fluorescence units; fluor., fluorescence; conc., concentration; min, minutes.