Anti cd3 fitc

To assess the quality of T cell isolation, cells were fluorescently stained with anti-CD45-V500, anti-CD3-FITC, anti-CD4-APC-H7, anti-CD8a-PE, anti-CD19-APC, anti-CD14-PerCP, and anti-CD56-PE-Cy7 antibodies (Beckton Dickinson, Franklin Lakes, USA). For monitoring T cell subsets (day 3 to day 8), cells were fluorescently stained with anti-CD3-FITC, anti-CD4-BV510, anti-CD8-PE-Cy7, anti-CD127-AlexaFluor®647, anti-CD25-PE, anti-CD69-APC-H7, and anti-LAG-3-BV421 antibodies (Beckton Dickinson, Franklin Lakes, USA). Briefly, cells were harvested, washed, resuspended in 1× phosphate buffered saline (PBS) at pH 7.4, and incubated with antibodies for 30 min at 4 °C. After antibody incubation, cells were washed with PBS, incubated with 7-aminoactinomycin (7-AAD) reagent 10 min prior to measurement and analyzed via flow cytometry as described below.
All cells were analyzed by flow cytometry using BD FACSCanto™ II (Beckton Dickinson, Franklin Lakes, USA). Dead cells and cell debris were excluded from the analysis based on 7-AAD fluorescence and scatter signals. Analysis was performed using Kaluza flow cytometry analysis software (Beckman Coulter, Brea, USA).

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.