These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

Recent data from murine models have confirmed that Langerhans cells are not the only population of APCs in the skin involved in initiating immune responses. In healthy human skin, we identify CD1a⁺ dermal APCs located close to the lymphatic vessels in the upper layers of the dermis that are unequivocally distinct from migrating Langerhans cells but exhibit both potent allostimulatory capacity and a chemotactic response to CCR7 ligands. In contrast, CD14⁺ dermal APCs are distributed throughout the dermis and lack a chemotactic response to CCR7 ligands. CD1a⁺ dermal APCs therefore represent an APC population distinct from Langerhans cells that are capable of migrating to lymph nodes and stimulating naive T cells. In humans, CD1a⁺ dermal APCs may fulfill some of the roles previously ascribed to Langerhans cells.

Nonstructural protein 4 (NSP4), encoded by rotavirus, exhibits various properties linked to viral pathogenesis, including enterotoxic activity. A recent study (O. V. Kavanagh et al., Vaccine 28:3106-3111, 2010) indicated that NSP4 also has adjuvant properties, suggesting a possible role in the innate immune response to rotavirus infection. We report here that NSP4 purified from the medium of rotavirus-infected Caco-2 cells triggers the secretion of proinflammatory cytokines from macrophage-like THP-1 cells and nitric oxide from murine RAW 264.7 cells. Secretion is accompanied by the stimulation of p38 and JNK mitogen-activated protein kinases (MAPKs) and nuclear factor NF-κB. NSP4 triggered the secretion of cytokines from murine macrophages derived from wild-type but not MyD88 ^−/− or Toll-like receptor 2 (TLR2 ^−/− ) mice and induced secretion of interleukin-8 (IL-8) from human embryonic kidney cells transfected with TLR2 but not TLR4. Our studies identify NSP4 as a pathogen-associated molecular pattern (PAMP) encoded by rotavirus and provide a mechanism for the production of proinflammatory cytokines associated with the clinical symptoms of infection in humans and animals.

Objectives: Trichomoniasis is a common sexually transmitted disease, and adhesion of the pathogenTrichomonas vaginalisto the host vaginal cells is the first step in establishing infection. For this to happen, the pathogen has to overcome a natural protective barrier composed mostly of lactobacilli. The objective of this study was to understand the role of lactobacilli in the adhesion ofT vaginalisto host cells.Methods: Adhesion assays were carried out by incubating vaginal epithelial cells (VECs) withT vaginalisand lactobacilli together and compared with non-lactobacilli recipient controls. By varying incubation parameters and testing several microbial isolates, the number of pathogens that adhered to the VECs was determined by flow cytometry.Results: Overall, but with few exceptions, lactobacilli caused inhibition ofT vaginalisadhesion to a variable degree.Lactobacillus gasseriATCC 9857 and CBI3 (ambiguousLactobacillus plantarumorLactobacillus pentosus) caused the highest level of parasite adhesion inhibition and enhancement, respectively. These isolates ofLactobacilluscan profoundly alter the adhesive properties of low-adherent and high-adherent strains ofT vaginalisin a dose-dependent manner. Additionally, the effects of lactobacilli onT vaginalisadhesion are strictly contact-dependent, and surface lipoglycans ofT vaginalisare most likely not involved in this modulation of adhesion mediated by the bacteria.Conclusions: Lactobacilli can modulate adhesion ofT vaginalisby significantly modifying the natural adhesive properties of variousT vaginalisstrains. This study highlights the importance of considering the role of the vaginal microbiota in the pathogenesis of trichomoniasis.