Collagenase 3

The spleen, tumor or tumor-draining lymph node (TDLN) cell isolation procedure was based on our previously described procedure55 (link). Tissues were dissected from freshly killed mice and trimmed of fat and connective tissue. Small cuts into the capsules were made with a pair of fine scissors, and the fragments were incubated in 1 mg/ml collagenase III (Gibco-Invitrogen) in RPMI 1640 medium at 37 °C for 15 min with gentle agitation using a Pasteur pipette every 5 min. Cells recovered by centrifugation were resuspended in FACS staining buffer (BD Pharmingen) and then passed through a 100-μm mesh strainer before counting and staining.

Cell culture materials were from Lonza, (Basel, CH) unless otherwise specified.
The pBC cell line was generated from a specimen of human invasive ductal breast carcinoma, derived by the surgeons of the Breast Unit at the University Hospital of Pisa. This breast carcinoma was previously shown to contain MMTV-env sequences using nested PCR [4 (link)]. The specimen was dissected under sterile conditions by an experienced pathologist and only tumoral pieces were selected. Tissue fragments were carefully minced into 2-3 mm cubes in a small volume of growth medium and washed with a mixture of PBS (Phosphate Buffered Saline), penicillin/streptomycin and fungizone.
The tissue fragments were incubated overnight at 37°C under 5% CO₂ with DMEM F12, 10% FBS (Fetal Bovine Serum) and 10 mg collagenase III (Invitrogen, Life Technologies). On the following day, the tissue fragments were centrifuged at 1200 rpm for 5 minutes and plated in a T25 flask with growth medium containing: DMEM F12, 1% penicillin/streptomycin, 1% hydrocortisone (Sigma), 1% fungizone, 5% FBS, 20 ng/ml hEGF (Human Epidermal Growth Factor, Miltenyi Biotec), 2% B27, 25ul Coleric Toxin (Reagent Proteins), 2ml Bovine Pituitary Extract (Invitrogen, Life Technologies).

The spleen, tumor or tumor-draining lymph node (TDLN) cell isolation procedure was based on our previously described protocol55 (link). Tissues were cut into fragments and then incubated in 1 mg/ml collagenase III (Gibco-Invitrogen) in RPMI 1640 at 37 °C for 15 min. Flow cytometry staining was performed using fluorescence-conjugated anti–mouse antibodies targeting CD4, CD8a, CD69, CD25, IFN-γ, IL17A, CD11b and CD11c (BD Pharmingen). Cells were also incubated with goat anti-Nrp-1 (R&D Systems) and rat anti-TGF-β1 (Biolegend) followed by an Alex Fluor 647-conjugated anti-goat antibody (BD Pharmingen) or Alex Fluor 488-conjugated anti-rat antibody (BD Pharmingen). Intracellular staining was performed using a Foxp3 staining kit and fixation/permeabilization buffer (eBioscience). Samples were obtained using a FACSCalibur equipped with Cell Quest software (BD Biosciences). The cells were gated for lymphocytes based on the forward and side scatter profiles. All data were analyzed using Tree Star FlowJo software.