Isolation of myeloid dendritic cells and epithelial cells from human thymus
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Isolation of myeloid dendritic cells and epithelial cells from human thymus. / Stoeckle, Christina; Rota, Ioanna A; Tolosa, Eva; Haller, Christoph; Melms, Arthur; Adamopoulou, Eleni.
in: JOVE-J VIS EXP, Nr. 79, 01.01.2013, S. e50951.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Isolation of myeloid dendritic cells and epithelial cells from human thymus
AU - Stoeckle, Christina
AU - Rota, Ioanna A
AU - Tolosa, Eva
AU - Haller, Christoph
AU - Melms, Arthur
AU - Adamopoulou, Eleni
PY - 2013/1/1
Y1 - 2013/1/1
N2 - In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c(+)), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45(hi)) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.
AB - In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c(+)), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45(hi)) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.
KW - Centrifugation, Density Gradient
KW - Cytological Techniques
KW - Dendritic Cells
KW - Epithelial Cells
KW - Humans
KW - Immunomagnetic Separation
KW - Myeloid Cells
KW - Thymus Gland
U2 - 10.3791/50951
DO - 10.3791/50951
M3 - SCORING: Journal article
C2 - 24084687
SP - e50951
JO - JOVE-J VIS EXP
JF - JOVE-J VIS EXP
SN - 1940-087X
IS - 79
ER -
