Murine gm csf

The femur and tibia from two 6-8 week old C57BL/6 or BALB/c mice (Harlan, Rossdorf, Germany) per experiment were flushed with medium (RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin, 50 µg/ml streptomycin, 100 µg/ml gentamycin; Gibco, Grand Island, New York, USA) to collect bone marrow cells. Erythrocytes were lysed in 150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, Steinheim, Germany), pH 7.2. The remaining bone marrow cells were seeded at 10⁶ cells/ml in medium supplemented with either 5 ng/ml murine GM-CSF (BD Pharmingen, Franklin Lakes, New Jersey, USA) and cultured for 7 days or with 100 ng/ml murine Flt3l (BD Pharmingen, Franklin Lakes, New Jersey, USA) and cultured for 9 days or with 10 ng/ml murine M-CSF (eBioscience Inc., San Diego, California, USA) and cultured for 7 days at 37°C with 5% CO₂ in a humidified atmosphere. The bone marrow-derived cells cultured in the presence of GM-CSF give rise to non-adherent in vitro conventional DC model populations, positive for the marker CD11c. Flt3l promotes the differentiation into a mixed population of in vitro model conventional DCs and plasmacytoid DCs, both positive for CD11c with the plasmacytoid DCs being additionally positive for B220, while the in vitro model MΦs cultured in the presence of M-CSF are positive for CD11b [41] (link)-[43] .

DCs were derived from bone marrow cells [31] (link). Cells were resuspended in Iscove’s Modified Dulbecco’s Medium (IMDM from Gibco, Invitrogen) supplemented with 10% FCS (PAA, Laboratories GmbH, Pasching, Austria), 1 mM sodium pyruvate, 1 mM non-essential amino acids, 100 U/ml penicillin/streptomycin (Gibco), 50 µM β-mercaptoethanol (Sigma-Aldrich), 5 ng/ml recombinant murine IL-4 (eBioscence) and 3 ng/ml murine GM-CSF (BD, Pharmingen) and incubated at 37°C and 5% CO₂ for 6 to 7 days.

Mice femur and tibial bones from 6–8-week-old C57BL/6 (Envigo, Borchen, Germany) were flushed with culture medium (RPMI 1640 supplemented with 10% fetal calf serum, 100 U/mL penicillin, 50 µg/mL streptomycin, 100 µg/mL gentamycin; Gibco, Paisley, UK) to collect bone marrow cells. Erythrocytes were lysed in ACK lysing buffer (Ammonium-Chloride-Potassium, Sigma-Aldrich, Darmstadt, Germany). Bone marrow cells were then seeded at 1.10⁶ cells/mL in culture medium supplemented with 5 ng/mL murine GM-CSF (BD Biosciences, San Diego, CA, USA) and cultured for 7 days.