Model elwd 0

The percentage of viable cells was determined by staining with 3,3 dihexyloxocarbocyanine iodide (DiOC6; Molecular Probes) and propidium iodide (PI; Molecular Probes), as previously described [38 (link)]. Relative changes in cell numbers were measured by counting cells at high sample flow for 20 seconds on a BD FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). The viability of CD3⁺ T cells was measured in CLL PBMC co-cultures with NLC by flow cytometry after staining with CD3-APC antibody (BD Pharmingen), Annexin-V-FITC, and 7-AAD (Biolegend). To assess the NLC numbers, nonadherent cells were removed by pipetting, the adherent NLC were fixed with absolute methanol for 5 minutes and then stained with modified Giemsa stain (diluted 1:20 in PBS) for 1 hour. NLC were visualized using a phase-contrast microscope (Model ELWD 0.3; Nikon, Melville, NY, USA) with a Ph1 Plan 10 DL/0.25 160/- objective lens. Images were captured with a Nikon D40 digital camera (Nikon Corp) with the help of digiCamControl ver. 1.2.0.0 (http://digicamcontrol.com/). Average numbers of NLC counted in 8 different visual fields are reported.