Il6

Common sources

Known influences with in vitro immune responses

Activated monocytic cells, fibroblasts, endothelial cells

Activated TH lymphocytes Activated TH lymphocytes

Activated TH lymphocytes

Activated monocytic cells, TH lymphocytes

Interferon y Activated monocytic cells, TH lymphocytes

Cofactor for T lymphocytes and antigen-stimulated B

lymphocytes Differentiation factor for antigen-stimulated B

lymphocytes; T lymphocyte growth factor Cofactor for T lymphocytes; differentiation factor for B lymphocytes (important in isotype switching); enhances APC activity Differentiation factor for B lymphocytes (important in isotype switching); cofactor with IL-2 for B lymphocytes Cofactor for B and T lymphocytes; differentiation factor for B lymphocytes (differentiation into plasma cells)

Cofactor with IL-2 for antibody secretion by B lymphocytes; regulator of T lymphocyte growth conjugate; separation from unlabeled cells employs a magnet or a fluorescence-activated electrical field. Examples of markers commonly used are CD14 on myeloid cells (not all dendritic cells are CD14+), surface immunoglobulin on B cells, and CD2, CD4 or

CD8 on T lymphocytes (the latter two will be useful for separating T lymphocyte subpopulations); the T lymphocyte CD3 is not employed, because antibody reaction with this marker can stimulate the cells. An additional modification to the procedure employs

CD106 L-seledin llgand

Figure 3 Accessory endothelial cell interaction with T lymphocytes and monocytic APC, via adhesion molecules and their ligands

CD106 L-seledin llgand

Aecessoiy oclls

Figure 3 Accessory endothelial cell interaction with T lymphocytes and monocytic APC, via adhesion molecules and their ligands

Secondary lymplxnd ong.'in (lymph node, spleen. lonslls)

Cut organ into pieces < 3 mm square

Pelfl deh 01

llnsls

Vononudcar etil suspension plastic adherence + low density

Colla genasC ONA use di gcslion iQQQ Dendritic cell APC

Colla gemsf ONAjäse <|i^esiion

Lyse erythrocytes (if necessary) M_

Wash

Resuspend in required medium with lflii hornotogoiisseram

Monocyte cell APC

Percherai Wood

Gulfy coats

Fieoll-Dialrizoale densHy ccnlfifugBlion

Mononaclea* col suspension plastic adherence + low density

Resuspend in required medium with lflii hornotogoiisseram

Mononaclea* col suspension

iQQQ Dendritic cell APC

Monocyte cell APC

mononuclear cell cultures

Figure 4 Summary of the methods employed for the preparation of mononuclear cells from lymphoid organs and the blood. These sources of mononuclear cells can be used for the preparation of APCs, or for the cultures to be stimulated by antigen in the immune response in vitro.

mononuclear cell cultures

Figure 4 Summary of the methods employed for the preparation of mononuclear cells from lymphoid organs and the blood. These sources of mononuclear cells can be used for the preparation of APCs, or for the cultures to be stimulated by antigen in the immune response in vitro.

Secondary lymphoid organ (lymph node, spleen, tonsils)

Sûconda/y lymphoid organ (lymph node, spleen, tonsils)

Umblbcal coid, skin, lungs

Secondary lymphoid organ (lymph node, spleen, tonsils)

Mononucl«ar tell tu Sponsion

Pro-formed monJayûr of accessory cells cocuhure mononuclear cells +

accessary eels

Antigen

woll'piato culura or llttik eullure

Figure 5 Summary of the preparation of preformed monolayers of accessory cells, and their application to coculture with mononuclear cells in immune response systems in vitro.

complement-mediated lysis to deplete unwanted cells: the antibody-labeled cells are treated with rabbit anti-mouse immunoglobulin and complement to induce the lysis.

A decision about whether or not to employ enrichment will depend on the required use of the in vitro immune response. Enrichment is not always essential. Its application should be carefully considered in the context of how beneficial it would be to the objectives of the experimentation.

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