CCL21 is retained on cell membrane and not detected in the supernatant. (A) Image galleries of single macrophages (as defined in panel 3A) showing CCL21 (green), CD163 (yellow), CCL21 mRNA (red), and CD68 (cyan) fluorescent signals and Brightfield (BF) in permeabilized (upper and middle) and nonpermeabilized (lower) conditions in presence (upper) or absence (middle and lower) of BFA treatment. (B) Confocal images of adherent CD68⁺ cells stained with anti-CCL21 Ab in permeabilized (left) or nonpermeabilized (right) conditions (objective ×63; scale bar represents 12 μm). (C) CCL21, CXCL13, and CXCL12 protein levels secreted by adherent NLCs after 14 days of culture detected by Luminex technology. Dots represent individual protein levels from 14 patients with CLL. (D) Detection of CXCL13 mRNA in single macrophages. Image galleries of single macrophages showing RPL13 mRNA (green), CD163 (yellow), CXCL13 mRNA (red), and CD68 (cyan) fluorescent signals and Brightfield (BF). RPL13 mRNA of ribosomal protein L13A was used as housekeeping control. (E) Image gallery of aggregates showing macrophages and B cells based on CD206 (green) and CD68 (cyan) or CCR7 (yellow) and CD19 (red) fluorescent signals, respectively, as well as Brightfield (BF) in permeabilized conditions. Arrows show B cells present in the aggregates. (F) Graph shows the CCR7 MFI (Mean Fluorescence Intensity) values detected on B cells in suspension or remaining in the adherent fraction after 14 days of culture.