Generation of Bone Marrow-Derived Dendritic Cells

BMDCs were generated as previously described (25 (link), 30 (link), 31 (link), 33 (link), 34 (link)). Briefly, femurs and tibiae were collected from freshly euthanized B6 mice, BM harvested, and single cell seeded at 2 × 10⁵/ml. RPMI 1640 (Lonza) was supplemented with 10% FBS and 1% penicillin/streptomycin, plus 20 ng/ml mouse GM-CSF (all from BioLegend). For generation of CD103⁺ BMDCs, Flt-3 (100 ng/ml, eBioscience) was used instead of GM-CSF. On day 4, fresh GM-CSF (20 ng/ml) was added to both culture conditions.

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Ahadome S.D., Mathew R., Reyes N.J., Mettu P.S., Cousins S.W., Calder V.L, & Saban D.R. (2016). Classical dendritic cells mediate fibrosis directly via the retinoic acid pathway in severe eye allergy. JCI Insight, 1(12), e87012.

Publication 2016

RPMI 1640 mouse GM-CSF Flt-3 GM-CSF

Cd103 Cell Femurs Flt 3 Gm csf Mouse Penicillin Streptomycin Tibiae

Corresponding Organization : Duke University

Other organizations : University College London

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Variable analysis

independent variables

Generation of BMDCs: GM-CSF (20 ng/ml) or Flt-3 (100 ng/ml)

dependent variables

Differentiation and generation of CD103+ BMDCs

control variables

Mouse strain: B6 mice
Cell seeding density: 2 × 10^5/ml
Culture medium: RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin
Timing: Fresh GM-CSF (20 ng/ml) added on day 4 for both culture conditions

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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