Annexinv pacific blue

HG3 and MEC-1 CLL cell lines (200 000 cells/mL) and primary CLL cells (2 × 10⁶ cells/mL) with > 90% tumor B cells, were incubated alone or in coculture with HS-5 cells (50,000 cells/mL) for 48 h with the compounds at doses ranging from 0.1 to 15 µM. Cell cytotoxicity was quantified by double staining with Annexin-V conjugated to fluorescein isothiocyanate (FITC) and propidium iodide (PI) (eBiosciences, San Diego). Normal PBMCs were labeled simultaneously with anti-CD3-FITC, anti-CD19-Phycoeritrin (PE; Becton Dickinson, Franklin Lakes, NJ, USA) antibodies, and Annexin V-Pacific Blue (Life Technologies). Labeled samples were analyzed on an Attune focusing acoustic cytometer (Life Technologies). Cytotoxicity values were represented relative to untreated control. For analysis of cell viability due to an effect on cell proliferation and/or cell death , cells were cultured with the drugs for 48 h and 0.5 mg/mL MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) reagent (Sigma-Aldrich, St Louis, MO) was added for 2–6 additional hours before spectrophotometric measurement. Each measurement was made in triplicate. Values were represented using untreated control cells as reference.

The WST-1 reagent from Roche was used to determine cell viability, while apoptosis was determined by Annexin V Pacific Blue and TO-PRO-3 (Life Technologies; Grand Island, NY) staining, along with Count Bright Beads (Thermo Fisher Scientific), and analyzed by flow using FlowJo, version 10 (Tree Star, Inc.; Ashland, OR). Cell cycle analysis was performed using the NPE Analyzer^TM reagent (NPE Systems, Inc.; Pembrook Pines, FL) on a Gallios flow cytometer (Beckman Coulter, Inc.; Brea, CA), and analyzed using Multicycle Software (Phoenix Flow Systems; San Diego, CA). Live cells were treated with 1 μM ER-Tracker Green (Thermo Fisher Scientific) for 1 h at 37°C and washed in PBS prior to analysis with flow using the FITC channel.