Langerhans cell (LC) activation in relation to the interval between primary melanoma removal and sentinel lymph node (SLN) excision and Breslow thickness. Human SLN single-cell suspensions from early-stage melanoma patients (SLN⁻, n = 14) were obtained after informed consent in accordance with the Declaration of Helsinki and phenotypically analyzed by flow cytometry using (combinations of) the following monoclonal antibodies diluted in PBS supplemented with 0.1% BSA and 0.02% NaN₃ (FACS buffer) and incubated for 30 minutes at 4°C: CD11c-APC, CD14-PerCP_Cy5, CD1a-PE, CD1a-FITC, CD80-FITC, CD86-FITC (BD Biosciences), Langerin-PE (intracellular staining by use of the BD Fix-Perm kit), and CD83-FITC (Beckman Coulter Immunotech). After incubation, cells were washed in FACS buffer to remove excess antibodies. Cells (0.25-0.5 × 10⁶) were analyzed on a FACSCalibur flow cytometer (BD Biosciences) equipped with CellQuest Pro 6.0 analysis software.⁴  Correlations between percentage of CD83⁺ LCs in SLNs and (A) excision interval (in months) and (B) Breslow thickness (in mm) were determined using the Pearson r test. Differences were considered significant when P < .05; 95% confidence intervals are depicted. (C) CD83, CD80, and CD86 expression levels among LCs (by percentage positive cells), compared among patients with relatively small (Breslow < 1.5 mm) and large (Breslow > 1.5 mm) primary tumors and excision intervals < 44 days. Differences were considered significant when P < .05 in an unpaired 2-sided Student t test.