CD68⁺ NLCs produce CCL21. (A) Assay outline and gating strategy applied to identify single cells and aggregates. Cells in camera focus were selected from all events on the basis of gradient RMS of the bright field image. Single B cells, macrophages, and cellular aggregates were identified by plotting “area” vs “aspect ratio” in which events with higher aspect ratio are assigned to single cells while those with lower aspect ratio are aggregates. (B) Image gallery of single macrophages showing different subpopulations based on CD14 (purple), CD206 (green), CD163 (yellow), CD19 (red), and CD68 (cyan) fluorescent signals as well as Brightfield (BF). (C) Detection of CCL21 mRNA and protein in CD68⁺ CD163⁺ or CD68⁺ CD163⁻ subpopulations. Galleries show CCL21 protein (green), CD163 (yellow), CCL21 mRNA (red), and CD68 (cyan) fluorescent signals as well as Brightfield (BF). (D) Image gallery of aggregates of macrophages and B cells showing CCL21 (green), CD163 (yellow), CCL21 mRNA (red), and CD68 (cyan) fluorescent signals as well as Brightfield (BF). Arrows show B cells present in the aggregates. Merged images show that B cells are negative for CCL21 and CCL21 mRNA signals. All the experiments were performed in permeabilized cells after brefeldin (BFA) treatment.