A Method for Generation of Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells
Figure 8
Bone marrow-derived macrophage obtained from fresh or cryopreserved bone marrow cells respond similarly to the infection with Leishmania amazonensis.
Bone marrow-derived macrophages (BMDM) obtained from fresh or cryopreserved (frozen) bone marrow cells were infected with L. amazonensis promastigotes constitutively expressing the GFP at an MOI of 10 parasites per cell. After 48 h and 96 h of infection, the percentages of L. amazonensis-GFP+ and propidium iodide-positive cells were estimated by flow cytometry. (A) Percentage of GFP+ BMDM obtained from fresh (white bars) and frozen (black bars) BM cells. (B) Mean fluorescence intensities (MIF) for GFP signal. (C) Representative images of cultures infected for 48 h e 96 h with L. amazonensis GFP. (D) FACS profiles of BMDM obtained from fresh and frozen BM cells infected with L. amazonensis GFP. Shown are the percentages of cells with signal for GFP and PI. (E) Bar graphics indicating the percentage of propidium iodide-positive cells in uninfected cultures (control), cells treated with 0.05% Tween 20 (Tween) or after infection with L. amazonensis for 48 and 96 hs. Data are representative of those found in three independent experiments. No statistically significant differences were detected between BMDM obtained from fresh or frozen BM cells (P>0.05).