Mus musculus
source_name
Bone marow dendritic cells
cell type
Dendritic cells from bone marrow
strain
C57BL/6
growth protocol
Bone marrow cells from the tibiae and femurs of 8-week-old male mouse were flushed with PBS with 0.5mM EDTA (Thermo Fisher Scientific, MA, USA), and depleted of red blood cells with ACK (Ammonium-Chloride-Potassium) lysing buffer (Thermo Fisher Scientific, MA, USA). Cells were plated in a 10 ml dish (1x106 cells/mL) in RPMI-1640 (Thermo Fisher Scientific, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, MA, USA), 2mM L-glutamine, 1mM sodium pyruvate, 1 mM nonessential amino acids, 25 mM HEPES, 100 U/mL penicillin (Thermo Fisher Scientific, MA, USA), 100 mg/mL streptomycin (Thermo Fisher Scientific, MA, USA), 25 μg/mL rmGM-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany), and 25 μg/mL rmIL-4 (Miltenyi Biotec, Bergisch Gladbach, Germany) at 37°C in a humidified 5% CO2 atmosphere. On day 5 cells were harvested, restimulated with new growth factors and plated 1x106 cells/mL on 12-well culture plate.
treatment protocol
On day 8 BMDCs were stimulated with 1 μg/mL of LPS (Sigma-Aldrich, St Louis, MO, USA) for 6 hours.