Miltenyi s gentle dissociator

Whole mouse stomachs were harvested and processed using Miltenyi’s Gentle Dissociator (Miltenyi Biotec, Boston, MA). In brief, the stomach was cut into 5 mm pieces and then transferred to a C-tube (Miltenyi Biotec) in 5 ml RPMI/10% FBS. The preset Miltenyi Biotec program m_imptumor 02 was run once and then the tissue was digested for 30 min at 37°C in a solution containing (0.32 mg/ml Dispase and 0.30 mg/ml Collagenase D, Roche) while shaking in a CO2 incubator. After the 37°C incubation, 100U/ml of DNase (Sigma) was added to each tube and the Miltenyi Gentle Dissociator was run for a second and third time using the preset program m_imptumor 02. The tissue homogenate was passed through a 70 μm cell strainer (BD). Cells were harvested by centrifugation, washed, and then live cells were counted by using a hemocytometer and trypan blue exclusion staining. The samples were stained with 2 μg/ml anti- F4/80, 1.5 μg/ml anti-Gr1 (clone RB6-8C5), and 2 μg/ml anti-CD11b (clone M1/70) (all antibodies were purchased from BD Biosciences, and analyzed on a BD LSR II flow cytometer).