Hyclone l 15 leibowitz medium

After mice were euthanized, spleens and tumor tissues (if applicable) were collected in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS. Spleens were processed using a stomacher, red blood cells were lysed using ACK lysis buffer (LifeTechnologies), and the remaining cells were filtered through a 40μm filter. Tumors were minced using a scalpel, and incubated in a tumor dissociation enzyme mix consisting of: 170mg/L Collagenase I, II and IV (ThermoFisher), 12.5mg/L DNAse I (Roche), 25mg/L Elastase (Worthington) in 50% RPMI + 10% FBS and 50% Hyclone L-15 Leibowitz medium (ThermoFisher). Tumors were incubated in this mixture with end-over-end mixing for 1 hour at 37°C, and then filtered twice through a 40μm filter prior to plating for staining.

Spleens from immunized mice were harvested in RPMI medium supplemented with 10% FBS. Splenocytes were dissociated using a stomacher, filtered and Red Blood Cells were lysed using ACK Lysis Buffer (LifeTechnologies). Cells were filtered through a 40μm filter, and counted and plated for staining or for ELISpots. Tumors were mechanically dissociated using a scalpel, and then incubated in a mixture of Collagenase I, II and IV (170mg/L, ThermoFisher), DNAseI (12.5mg/L, Roche), Elastase (25mg/L, Worthington) in a 50/50 mixture of Hyclone L-15 Leibowitz medium (ThermoFisher) and RPMI + 10% FBS + 1% Penicillin/Streptomycin. Dissociated cells were then filtered twice through a 40μm filter, and plated for stimulation and staining.