Cytomic fc 500mcl flow cytometer

Mice bearing melanoma allografts were i.p. injected with PBS, VNP20009 and two flagellum-deficient strains. The tumors and spleens were resected after 6 days. Half of the tumor mass was dissected and incubated in PBS containing collagenase I (Gibco), collagenase IV (Sigma–Aldrich), DNase I (Sigma–Aldrich) and hyaluronidase (Worthington) for 1 h at 37 °C. Then the cell suspension was filtered through a 70 μm cell strainer to form single cell suspension. Then red blood cells were removed by 5 min of incubation with red blood cell lysis buffer on ice. The mice spleens were smashed thoroughly between frosted glass slides and filtered through a 70 μm cell strainer to prepare single cell suspension. Then red blood cells were removed by 5 min of incubation with red blood cell lysis buffer on ice.
After that, cells were stained with following antibodies: anti-CD4-PE (#553652, BD Pharmingen), anti-CD8-FITC (#561966, BD Pharmingen), anti-F4/80-PE (#565410, BD Pharmingen) and were processed for flow cytometry analysis by Cytomic FC 500MCL flow cytometer (Beckman Coulter).