Abstract
Dendritic cell (DC) maturation approved to be a pivotal process for initiating immunity. Many protocols were established in order the isolated peripheral blood mononuclear cells (PBMCs) from healthy donors to mature into dendritic cells (DCs). The purpose of this study was to present an effective and reliable DC maturation procedure by comparing three different protocols (Interleukin-4/Tumor Necrosis Factor-appha (IL-4/TNFa) DC protocol, Interferon alpha (IFNa) DC protocol and FAST DC protocol). Whole blood was collected from six healthy donors and PBMCs were isolated by Ficoll gradient centrifugation. The counted cells were incubated with the addition of three different cocktails of supplements for appropriate time period. The final mature DC population was examined either by its phenotypic characteristics under light microscope or by measuring the expression of antigen presenting molecules such as CD80 and CD86 by flow cytometry. It was found that the mature DCs, generated from the IL-4/TNFa DC protocol, expressed higher levels of CD80 and CD86. Furthermore, they sharply exhibited their phenotypic hallmarks.
Highlights
Dendritic cell (DC) are exceptionally powerful initiators of immunity, with the ability to activate an immune response more potently than any other cell in the immune repertoire
The resulting immature DC were further treated by a 2 day culture step with fresh medium X-VIVO 20 medium containing TNFa and GM-CSF, 2) serumfree X-VIVO 20 medium supplemented with IFN-α and GM-CSF for 3 days and 3) RPMI 10% Fetal Bovine Serum (FBS) medium containing GM-CSF and IL-4 for 24 h followed by an another 24 h incubation with TNFa, Interleukin1 beta (IL-1β), Interleukin-6 (IL-6), prostaglandin 2 (PGE2) and CD40 ligand (CD40L)
The resulting immature DC were further treated by a 2 day culture step with fresh medium X-VIVO 20 medium containing 1000 U/ml TNF-α (300-01A, PeproTech) and 800 U/ml GM-CSF
Summary
DCs are exceptionally powerful initiators of immunity, with the ability to activate an immune response more potently than any other cell in the immune repertoire. Because of this unique hallmark, DCs are a major focus of laboratory and clinical study and are critical targets in cancer vaccine development [1] [2]. Cancer malignancies are often treated by administering a DC vaccine during the premalignant stages, prior to development of immune suppression [3]. As it is well-known, DCs are antigen-presenting cells of the mammalian immune system.
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