Macrophage cultures in vitro and transfer to EAN mice
After lysis of the red blood cells, the collected bone marrow cells from
femur and tibia were grown in complete RPMI-1640 medium (Gibco, Waltham,
MA) containing 10% Fetal bovine serum (FBS, Sigma Aldrich, St Louis,
MO), penicillin (100U/ml) and streptomycin (100U/ml).
1×106 cells was seeded into 96-well plates, then
stimulated with macrophage colony-stimulating factor (M-CSF) (10 ng/ml;
Pepro-Tech, Rocky Hill, NJ) for 48 hours (h), and afterwards LPS (for M1
macrophage) or IL-4 (for M2 macrophage) was added and incubated for
another 48h. Thereafter, cells were washed for three times by phosphate
buffer saline (PBS) to prepare for flow cytometry. The staining
procedure was similar to that of the spleen cells as mentioned above. A
part of cultured cells (M2 macrophage) stimulated with M-CSF and IL-4,
respectively were harvested and incubated with P0 (20 µg/ml) peptide for
4 h at 37°C. After washed with PBS twice, the cells were transferred
into EAN mice (0.1 ml, 1 × 106 cells for each mouse)
via the caudal vein.
The other cultured cells (M1 and M2 macrophage) were added by Bay11-7082
(1 µM) and incubated for 48h, then
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
solution and Dimethylsulfoxide (150 µl) was added to each hole.