Flow cytometry analyses of LCs in SLN from melanoma patients. SLN single cell suspension was obtained as described3 with modifications.4 Cells were collected in PBS, without enzymatic treatments or culturing, and immediately processed for flow cytometry analysis. Five-color cell staining was performed after cell membrane permeabilization with antibodies against Langerin PE (Immunotech), CD80 FITC and CD86 Pe-Cy5 (BD Pharmingen), CD83 APC and HLA-DR Pe-Cy7 (BioLegend). Cells were analyzed using FACS Canto with FACSDiva Version 6.0 software (BD Immunocytometry Systems). Isotype-matched Abs were used as negative controls. Results are expressed as percentages of double positive cells (left panels) and mean fluorescence intensity (right panels) among total gated Langerin+ cells, from freshly isolated and in vitro-migrated epidermal LCs (n = 5; from normal skin dermatomes of informed patients undergoing plastic surgery) and negative and positive SLNs LCs (n = 27 and n = 16, respectively). Bars represent mean value and asterisks indicate P value by 2-side Student t test statistical analysis (*P < .05; **P < .01; ***P < .001, respectively).
