NLC in vitro differentiation. Expression of (A) CD68, (B) vimentin, (C) CD163, and (D) CD14 of in vitro differentiated NLCs. Fresh CLL cells or CLL LN single cells were cultured in 4-well chambered slides for 1 to 2 weeks. Fresh CLL cell slides were fixed the day of separation from blood. Slides were costained with a rabbit anti-HMGB1 antibody (green) and DAPI. (E) Determination of NLC marker proteins by flow cytometry. CLL mononuclear cells were cultured for 2 weeks. Cells were fixed/permeabilized and then stained with anti–CD14-FITC, anti–CD68-PE, and anti–CD163-Allophycocyanin or relative isotype controls. The NLC population that is CD19-AF-488 negative and contains larger sizes (forward scatter) and high granulation (side scatter) was selected for marker protein analysis. Empty peaks were those stained with isotype controls and solid peaks were cells stained with specific antibodies. The flow cytometry profiles in the lower panel represent negative expression of 3 markers from selected small CD19-AF-488–positive CLL cells. Data shown are 1 of the typical CLL samples from 3 individual cases studied.