Flow cytometry fixation permeabilization buffer kit 1

Tumor masses were cut into pieces and digested into mononuclear cell suspension according to the protocol 31 (link). Then, mononuclear cell suspension was blocked with anti- mouse CD16/32 (BD Biosciences, San Jose, CA, USA), followed by incubating at 4 °C for 30 min with fluorescein-conjugated specific antibodies against surface antigens CD45 (#563891, BD Pharmingen, CA, USA), CD11b (#553310, BD Pharmingen), F4/80 (#565411, BD Pharmingen), MHCII (#557000, BD Pharmingen), CD206 (FAB25351R-100UG, R&D Systems), CD3e (#551163, BD Pharmingen), CD4 (#552051, BD Pharmingen), and CD8a (#553030, BD Pharmingen). Then, intracellular staining was performed using Flow Cytometry Fixation & Permeabilization Buffer Kit I (#FC009, R&D Systems). IFN-γ (#554412, BD Pharmingen) and TNF-α (#554420, BD Pharmingen) antibodies were used for intracellular staining. Matched non-specific isotype immunoglobulins were stained as controls. 7-ADD attaining was used to exclude dead cells. After washing twice with staining buffer, cells were resuspended in 300 μl of PBS with 1% FBS and analyzed using a BD LSRFortessa flow cytometer (BD Biosciences). Results were processed and visualized with FlowJo V10 software (TreeStar, Ashland, OR, USA).

The cells grown in 3D matrigel were dissociated with dispase (17105041 ThermoFisher) at 37 °C for 1 hour centrifuged at 300 g × 5 min and washed in 1x cold PBS. To generate single cell suspensions, cell pellets were incubated in TrypLE for 10–15 minutes. The reaction was diluted in DMEM/F12 (Life Technologies) with 2% (v/v) FBS and centrifuged at 300 g at room temperature. The cell pellets were immersed with sorting buffer 3% (v/v) FBS in PBS, cell aggregates were removed using a cell strainer with a 70-µm pore size (BD falcon) and single cells were collected. For surface antigens, the cells were incubated with primary antibodies for 30 minutes with gentle shaking, washed twice with 3% (v/v) FBS/PBS, and if necessary, incubated with the secondary antibodies for 30 minutes followed by two more rinses with 3% (v/v) FBS/PBS. For intracellular antigens, the Flow Cytometry Fixation & Permeabilization Buffer Kit I, Flow Cytometry Permeabilization/Wash Buffer I and Flow Cytometry Staining Buffer kits (R&D). The cells were analyzed using a BD FACS Aria II flow cytometer (BD Biosciences). Unstained controls were used for gating conjugated antibodies and secondary only controls were used for gating unconjugated antibodies. The list of antibodies used for FACS is shown in Supplementary Table S2.