Human Flt3L Expands Dog DCs Figure 6. Allogeneic mixed-lymphocyte reaction (MLR). DCs (stimulators, A) from dog peripheral blood monocytes cultured with either Ad.hFlt3L conditioned media (purple dots) or IL-4+GM-CSF (orange dots) were incubated with allogeneic spleen T cells (responders, B) purified by Fluorescence Activated Cell Sorting (FACS) from the spleen of a second dog. Dot plots show the percentage of CD11c+-cells (A) and the percentage of CD8+ and CD4+ T cells collected from dog spleen by FACS (B). SSC: Side Scatter; FSC: Forward Scatter. C, T cell proliferation was determined by BrdU incorporation after 4 days in co-culture. Additional controls included DCs incubated without T cells (broken lines). *p,0.05 vs. DCs no T cells (Randomization test). doi:10.1371/journal.pone.0011074.g006 Allogeneic mixed leukocyte reaction (MLR) After 7 days culture in hsFlt3L or canine IL-4/GM-CSF, non-adherent PBDCs were harvested and incubated 6 h with 50 mg/ml CpG2007 to be used as stimulators in an alloegeneic MLR. Dog spleen was harvested as described above from a second dog and 1 inch3 cubes were dissected and homogenized to release splenocytes. Red blood cells were removed by incubating in 3 volumes of ACK solution (0.15 mM NH4Cl, 10 mM KHCO3, and 0.1 mM sodium EDTA at pH 7.2) for 2 min and then washed with supplemented RPMI. Cells were passed trough a cell strainer BD Pharmingen 35-2350) to discard large debris. Splenocytes were frozen in 10% DMSO-90% FBS in liquid nitrogen until used for the allogeneic MLR. Dog splenocytes were thawed and stained with 8 mg/ml mouse anti-canine CD4 and CD8 (generated in the PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e11074