My normal protocol is Flt3L (100ng/ml) supplemented culture media (10% FCS, PSG and 2me in RPMI) driven for 7 days (half the media replenished at day 3 or 4).
Any reference to a better method or advice on ways to improve the number of pDCs I get would be greatly appreciated!
Take a look at the following protocol
Nature 434, 1035-1040 (21 April 2005) | doi:10.1038/nature03547; Received 18 February 2005; Accepted 14 March 2005; Published online 6 April 2005
Spatiotemporal regulation of MyD88–IRF-7 signalling for robust type-I interferon induction
Kenya Honda1,4, Yusuke Ohba1,2,4, Hideyuki Yanai1,4, Hideo Negishi1, Tatsuaki Mizutani1, Akinori Takaoka1, Choji Taya3 & Tadatsugu Taniguchi1
Preparation of DCs
To prepare bone-marrow-derived DCs, bone marrow cells were cultured for 6 days with 100 ng ml 21 human Flt3L (Fms-like tyrosine kinase 3 ligand, PeproTech) in RPMI 1640 medium. Collected cells were incubated with the pDC-specific rat monoclonal antibody 120G8 (ref. 30; provided by G. Trinchieri) and anti-rat IgG microbeads (Miltenyi Biotec) and separated into pDCs (positively selected cells) and cDCs (flow-through cells) using a magnetic-activated cell sorting (MACS) column. In some experiments, collected cells were incubated with anti-B220 and anti-CD11c antibodies (BD Biosciences), and B220 2/CD11c+ cDCs and B220+/CD11cintermediate pDCs were sorted using FACS Diva (BD Biosciences). To prepare splenic pDCs, spleens were digested with collagenase A(Roche Biochemicals) and pDCs were purified using a pDC isolation kit (Miltenyi Biotec)according to the manufacturer’s instructions.
The 120G8 rat antibody that targets the BST2 molecule, was developed in 2000 by the team of G.Trinchieri. This antibody is the property of Schering Plough and has been licensed to DENDRITICS and is distributed in the UK by 2BScientific.
It is very commonly used by the scientific community for its ability to deplete in vivo murine pDCs.
This antibody is also regularly used to purify the spleen pDCs (MIPC) where it is more efficient than PDCA1 giving good yields and higher purity .However, 120G8 as PDCA1 can activate pDCs and it must not neglect its action: modification of the phenotype, effect on the secretion of IFN.a, induction of apoptosis ...
See: Asselin-Paturel et al.
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