Measurement, time course, and titration of CLL B-cell pseudoemperipolesis. (A) To measure pseudoemperipolesis, CLL cells that had migrated into the stromal cell layer were harvested by treating the washed stromal cell layer with trypsin. The removed cells were analyzed via flow cytometry. We collected the data on the cells that had characteristic forward- and side-light scatter characteristics of lymphocytes, allowing us to exclude the marrow stromal cells from the analyses (for demonstrative purposes, the stromal cell population was centered for the acquisition of this sample). (B) The time course of pseudoemperipolesis of CLL B cells from 3 different patients. A continuous increase of CLL B cells, as determined by counting and anti-CD19 staining of cells that had migrated into the stromal cell layer, was detected over the first 2 hours. (C) Titration of pseudoemperipolesis of CLL cells using increasing numbers of input CLL cells. Lymphocytes from 3 different CLL patients that migrated into the stromal cell layer within 2 hours were counted for 20 seconds at high flow using a lymphocyte gate. Displayed are the mean (±SD) relative numbers of duplicate samples.