PMC:4236617 / 17688-19246 JSONTXT

{"project":"2_test","denotations":[{"id":"25070625-14966192-12189976","span":{"begin":339,"end":341},"obj":"14966192"}],"text":"Isolation and generation of human monocyte-derived dendritic cells (moDCs)\nLeukocyte-rich buffy coats, donated by healthy volunteers, as well as the permission to use human leukocytes, were obtained from the Finnish Red Cross Blood Service. Monocytes were purified and cultured in vitro to generate moDCs using a method described earlier [37] with minor modifications. Briefly, peripheral blood mononuclear cells were first isolated by Ficoll-Paque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation using Leucosep separation tubes (Greiner Bio-One, Germany), followed by a Percoll (GE Healthcare, Little Chalfont, UK) gradient centrifugation step. After magnetic beading using anti-CD3 and anti-CD19 beads (Dynal Invitrogen, Life Technologies, Carlsbad, CA), monocytes were allowed to adhere to 24-well plates (Falcon, BD, Franklin Lakes, New Jersey) for 1 h in the presence of RPMI 1640 (Sigma) supplemented with 20 mM HEPES, penicillin and streptomycin (100 IU/ml) and 2 mM L-glutamine without serum. The adhering cells were washed twice with PBS, after which differentiation was induced by maintaining the cells in RPMI 1640 (supplemented as described above) containing 10% (v/v) FCS (Integro, Zaandam, the Netherlands), 10 ng/ml human recombinant granulocyte macrophage-colony stimulating factor (GM-CSF, Gibco Life Technologies, Carlsbad, CA), and 20 ng/ml human recombinant interleukin 4 (IL-4, Gibco Life Technologies, Carlsbad, CA). MoDCs were used on day 7 in the experiments. In each experiment, cells from four donors were used."}

{"project":"MicrobeTaxon","denotations":[{"id":"T109","span":{"begin":28,"end":33},"obj":"9606"},{"id":"T110","span":{"begin":167,"end":172},"obj":"9606"},{"id":"T111","span":{"begin":1252,"end":1257},"obj":"9606"},{"id":"T112","span":{"begin":1381,"end":1386},"obj":"9606"},{"id":"T113","span":{"begin":114,"end":132},"obj":"9606"}],"namespaces":[{"prefix":"_base","uri":"http://purl.bioontology.org/ontology/NCBITAXON/"}],"text":"Isolation and generation of human monocyte-derived dendritic cells (moDCs)\nLeukocyte-rich buffy coats, donated by healthy volunteers, as well as the permission to use human leukocytes, were obtained from the Finnish Red Cross Blood Service. Monocytes were purified and cultured in vitro to generate moDCs using a method described earlier [37] with minor modifications. Briefly, peripheral blood mononuclear cells were first isolated by Ficoll-Paque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation using Leucosep separation tubes (Greiner Bio-One, Germany), followed by a Percoll (GE Healthcare, Little Chalfont, UK) gradient centrifugation step. After magnetic beading using anti-CD3 and anti-CD19 beads (Dynal Invitrogen, Life Technologies, Carlsbad, CA), monocytes were allowed to adhere to 24-well plates (Falcon, BD, Franklin Lakes, New Jersey) for 1 h in the presence of RPMI 1640 (Sigma) supplemented with 20 mM HEPES, penicillin and streptomycin (100 IU/ml) and 2 mM L-glutamine without serum. The adhering cells were washed twice with PBS, after which differentiation was induced by maintaining the cells in RPMI 1640 (supplemented as described above) containing 10% (v/v) FCS (Integro, Zaandam, the Netherlands), 10 ng/ml human recombinant granulocyte macrophage-colony stimulating factor (GM-CSF, Gibco Life Technologies, Carlsbad, CA), and 20 ng/ml human recombinant interleukin 4 (IL-4, Gibco Life Technologies, Carlsbad, CA). MoDCs were used on day 7 in the experiments. In each experiment, cells from four donors were used."}