In Vivo Assays of Langerhans Cell Migration

Dearman, and Ian Kimber 1. Introduction The skin is an immunologically active tissue the integrated skin immune system comprises epidermal Langerhans cells (LC), dermal dendritic cells (DC), tissue macrophages, mast cells, and T lymphocytes in transit. Cutaneous immune function is orchestrated through the action of cytokines produced locally in the skin by LC, keratinocytes, and other cell types (1-3). Key cellular vectors of cutaneous immune and allergic responses...

Isolation of Dendritic Cells from Human Afferent Lymph

Brand, Nikhil Yawalkar, Robert E. Hunger, and Lasse R. Braathen The skin acts as a mechanical, physicochemical, and immunological control and defense system. The efficient operation of the skin immune system involves cytokine production and adhesion molecule expression by both infiltrating and resident cutaneous cells, and thus depends upon a system of cell homing, based on interactions between cell-surface properties and soluble mediators. Inflammatory skin diseases demonstrate...

Isolation of Human Skin Dendritic Cells by In Vitro Migration

Hoekstra, John S. du Pont, Robert W. Kreis, and Eduard W. A. Kamperdijk In the human skin, various types of antigen-presenting cells (APC) are present. In the epidermis, they are identified ultrastructurally as Langerhans cells (LC) by the presence of Birbeck granules. LC are considered to belong to the family of dendritic cells (DC) that are important for the initiation of immune responses (1). In the dermis, macrophages and DC are present (2,3). The...

Isolation of Mouse Spleen Dendritic Cells

Stagg, Fiona Burke, Suzanne Hill, and Stella C. Knight It is now over 20 years since dendritic cells (DC) were first identified in and isolated from the spleens of mice (1,2) and they continue to be a much-studied population. Only a small proportion of spleen cells are DC, but the large size of the organ means that useful numbers of DC can still be purified. In recent years the ability to grow cells with the phenotypic and functional properties of DC from bone marrow progenitors has...

Of In Vitro Cultured Human Dendritic Cells

Giancarlo Bianchi, Giovanna D'Amico, Silvano Sozzani, Alberto Mantovani, and Paola Allavena Dendritic cells (DC) are professional antigen-presenting cells (APC) that are believed to be indispensable to initiate a primary immune response (1,2). DC are migratory cells that exhibit complex trafficking properties in vivo, involving interaction with vascular and lymphatic endothelium and extracellular matrix (ECM). DC progenitors from the bone marrow enter the blood and seed nonlymphoid tissues. DC...

Stephen P Robinson md phd Andrew J Stagg phd

M E T H O D S I N M O L E C U L A R M E D I C I N E 69. Gene Therapy Protocols, 2nd ed., edited by Jeffrey R. Morgan, 2002 68. Molecular Analysis of Cancer, edited by Jacqueline Boultwood and Carrie Fidler, 2002 67. Meningococcal Disease Methods and Protocols, edited by Andrew J. Pollard and Martin C. J. Maiden, 2001 66. Meningococcal Vaccines Methods and Protocols, edited by Andrew J. Pollard and Martin C. J. Maiden, 2001 65. Nonvira Vectors for Gene Therapy Methods and Protocols, edited by...

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Typical cobblestone morphology of endothelial cells when grown to confluence in culture. The cells form tight junctions that will not allow the passage of liquid through the monolayer. 3.2. Isolation of Blood Dendritic Cells 1. Defibrinate 70 mL of peripheral blood by shaking with glass beads. 2. Dilute the blood with an equal volume of RPMI and layer over Ficoll-Paque at a ratio of 3 vol Ficoll-Paque to 4 vol of diluted blood. 3. Centrifuge at 400g for 30 min and then harvest the...

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The different steps in an immunolabeling. An immunolabeling is performed on a piece on parafilm, preferably on a dark surface for better visualization of the grids. (A) Grids present are 200 L drops of PBS 50 mM glycine, 5 L drops of antibody (note that each grid is incubated separately), and 200 L drops of PBS 0.1 BSA. (B) The last step in the immunolabeling procedure is the removal of methyl cellulose uranyl acetate from the grid with a stainless-steel loop. Fig. 5. The different...

Enrichment of Freshly Isolated Langerhans Cells

Complement-Mediated lysis (Thy-1 kill) Pre-enrichment of freshly isolated LC is achieved by depleting the majority of keratinocytes as well as dendritic epidermal T cells (19,20) by complement-mediated lysis (see Note 6). 1. In a 50 mL polypropylene tube, resuspend 100-150 x 106 epidermal cells (the expected yield from 60 ears) in 3 mL of hybridoma culture supernatant of mAb anti-Thy-1. 2. Add 10 mL of a sterile-filtered solution consisting of 8.5 mL cytotoxicity medium, 1 mL of...

Generation of Blood Derived Human Dendritic Cells for Antitumor Immunotherapy

Nestle 1. Introduction Dendritic cells (DC) are a family of bone-marrow-derived professional antigen presenting cells (APC) with sparse, but wide, tissue distribution (1). They are classified primarily based on their localization as interdigitating reticulum cells when present in lymphoid organs, as veiled cells when present in afferent lymph, as Langerhans cells when present in the epidermis, and as dermal dendritic cells when found in the dermis. Although DC are...

Generation of Mouse Dendritic Cell Lines

Stefania Citterio, Maria Rescigno, Maria Foti, Francesca Granucci, Malgosia Matyszak, Giampiero Girolomoni, Dendritic cells (DC) are now recognized as major players in the control of immune responses (1), since they direct both the quality and the extent of the adaptative response. Thus, DC represent a very appropriate means for the manipulation of harmful or protective immunity (2-4). As DC are present in both lymphoid and nonlymphoid tissues, but in relatively low numbers, it is difficult to...

Isolation of Dendritic Cells from Mouse Lymph Nodes

Lymph nodes are the primary sites of T-cell stimulation by dendritic cells (DC). After contact with antigens, DCs migrate to draining lymph nodes from the skin and other tissues (1-3). Investigation of the morphology and function of lymph node DCs may provide important information about the role of these cells in normal and pathological conditions. Therefore, lymph nodes are popular sites for the isolation of dendritic cells. Dendritic cells isolated from lymph nodes represent interdigitating...

Isolation of Human Lung Dendritic Cells

G. Havenith, and Robert H. J. Beelen In the lung several cell types are capable of presenting antigen to T cells. The dendritic cells (DC) are the most potent antigen-presenting cell. DC form a rare cell population in the lung and early studies were hampered because scarce cell populations are seldom easy to isolate. Besides, this cell is pheno-typically heterogeneous depending on its localization, differentiation, or activation status. This chapter will...

DC as Targets for Adenovirus Vectors

Theoretically, direct gene transfer to DC has advantages in generating CTL toward the protein coded by the transgene, in that the gene will be transferred to the DC nucleus, expressed, and presented in the context of class I MHC for the immune system to recognize as self or nonself (28,29). In this regard, there is emerging evidence that, when Ad vectors expressing heterologous transgenes are administered in vivo, the Ad functions as an adjuvant to enhance cellular immune responses against the...

Isolation of Human Tonsillar Dendritic Cells

Tonsils as a Source of Human Lymphoid Dendritic Cells Tonsillectomy remains a frequently performed operation in developed countries ensuring that tonsils are the most readily available source of human lymphoid tissue and an easily accessible source of dendritic cells (DC). Tonsil lymphoid tissue also provides a source of the different DC that are resident within the B- and T-cell microenvironments. Although an alternative model for follicular dendritic cell (FDC) ontogeny has been proposed...

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Identification of CD11c+ and CD11c- DC in peripheral blood. This FACS profile (A) shows freshly separated human PBMC labeled with PE-conjugated anti-CD3, CD14, CD16, and CD19 and Cy-Chrome-conjugated anti- HLA-DR. DC (shown in the R1 gated region) are identified by lack of labeling with the cocktail of PE conjugated antibodies and labeling for HLA-DR with the Cy-Chrome-conjugated antibody. The DC from R1 in (A) are differentiated into two subpopulations by labelling with a FITC-labeled...

Separation of PBMC Subpopulations

PBMC can be separated into different subpopulations (mainly monocytes and T cells) by magnetic cell sorting (MACS). The technique relies on the monoclonal-antibody-based separation of PBMC with colloidal microspheres using a magnet (32). 3.2.1. Labeling and Separation of PBMC 1. (Continued from step 10 of Subheading 3.1.) Resuspend 1 x 109 cells in 750 ML of separation buffer (PBS-HA-EDTA), add them to a 25-mL Sterilin tube, and put on ice. It is important to work with homogeneous cell...