Ccl 213

Daudi cells were obtained from American Type Culture Collection (ATCC, CCL-213™). Daudi cells were harvested and successively washed in Roswell Park Memorial Institute (RPMI) and Hank’s Balanced Salt Solution (HBSS) media. Cell count was adjusted to 2 × 10⁶ cells/mL in HBSS buffer. The Daudi cells (5 × 10⁴ cells) were incubated for an hour at 4 °C in the dark with the various ADCs diluted at 0, 1, 3, 10, 30, or 100 µg/mL in HBSS buffered either at pH 6 (with 2-(N-morpholino) ethanesulfonic acid (MES)) or 7. They were then washed with HBSS and immediately processed on a 2-laser-8-colour Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Ten thousand events were acquired per condition by flow cytometry in the Gallios List Mode Data Acquisition and Analysis Software 1.2. Raw data from all samples were analyzed using Kaluza Analysis Software v.1.1.11052.10190 (Beckman Coulter). A gate was set on living cells using the forward scatter–side scatter plot. The ratio of mean fluorescence at each concentration versus cells alone was used to establish the fluorescence curves. All flow cytometry experiments were performed three times independently.