Gentlemacs disssociator

A Tumor Dissociation Kit, gentleMACS C Tubes, gentleMACS Disssociator, and MACSmix Tube Rotator were obtained from Miltenyi Biotec (Auburn, CA, USA). A Mouse CD3⁺ T Cell Isolation Kit, Mouse CD8⁺ T Cell Isolation Kit, polystyrene round-bottom tube, and Magnet for column-free immunomagnetic separation were obtained from STEMCELL Technologies (Vancouver, BC, Canada). Dynabeads Mouse T-Activator CD3/CD28 was purchased from Life Technologies (Carlsbad, CA, USA). A Cell Counting Kit-8 was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Oxa was purchased from TargetMol (Wellesley, MA, USA). Mouse IL-2 ELISA was purchased from BD Biosciences (San Diego, CA, USA). Mouse GrB ELISA was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-CD8 antibody and anti-GrB antibody were obtained from Cell Signaling Technology (Danvers, MA, USA).

Tumors were minced into pieces and carefully homogenized mechanically. The tumor mix was split into 3 samples of 150 mg carefully weighted with a high precision scale. The first sample was processed according to manufacturer protocol (Tumor Dissociation Kit (human), GentleMACS Disssociator, Milteny Biotec). The second and third samples were incubated in basal medium for 1 h while shaking with Collagenase A (type IV from Clostridium histolyticum, Sigma-Aldrich) for sample 2 or with 50 µg/ml liberase TH (Roche) for sample 3. After digestion, the three samples were processed the same way to avoid any technical bias. Briefly, digestion was stopped with fetal bovine serum (FBS) and cells washed and spun three times. Viability assay was performed in triplicate with 20 μl of the cell solution according to CellTiter-Glo manufacturer protocol (Promega). Number of viable cells was assessed using Kova Glasstic Slide (Fischer Scientific) after trypan blue staining. Two different tumor samples originating from two different patients were used for the test. Three counts were performed by two different researchers. Viability and number of viable cells were normalized to 1 mg of tumor.