LL37 efficiently transports self-DNA into human monocytes. (A) Degree of association of Alexa⁴⁸⁸-labeled huDNA-LL37 complexes with human blood-derived immune cells (pDCs, monocytes, and myeloid DCs), and structural nonhematopoietic cells (fibroblasts, keratinocytes) at different time points (0, 1, 4, and 12 hours) after stimulation, assessed by flow cytometry. Alexa⁴⁸⁸-labeled huDNA alone showed minimal association with all cell types tested. Data are the mean ± SEM of 4 independent experiments. *P < .0001 (paired Student t test). (B) Representative FACS analysis of Alexa⁴⁸⁸ huDNA-LL37 complex association with human blood-derived immune cells (pDCs, monocytes, and myeloid DCs), and structural nonhematopoietic cells (fibroblasts, keratinocytes) 4 hours after stimulation is shown. (C) Intracellular localization of Alexa⁴⁸⁸ huDNA-LL37 complexes 4 and 24 hours after stimulation of human monocytes, assessed by confocal microscopy. Green represents Alexa⁴⁸⁸ huDNA; and red, surface staining of monocytes with Alexa⁶⁴⁷ HLA-DR. Bar represents 5 μm (D) Percentage of cytoplasmic distribution of Alexa⁴⁸⁸ huDNA-LL37 complexes 4 and 24 hours after stimulation of human pDCs, and monocytes. Data are representative of 3 independent experiments and are given as mean ± SEM of the percentage of cells with cytoplasmic DNA^Alexa distribution; at least 150 cells containing Alexa⁴⁸⁸ huDNA-LL37 complexes from 10 different fields were counted for each condition. *P < .0001 (paired Student t test).