Gentlemacs mouse tumor dissociation kit

Tumors were disaggregated using the gentleMACS™ Mouse Tumor Dissociation kit (Miltenyi Biotec). Tumor-draining lymph nodes (or matching inguinal lymph nodes in non-tumor bearing mice) were dissociated through a 70 µm nylon cell strainer. Cells were stained with a fixable viability dye (Thermo Fisher) and blocked with antibodies against murine CD16/CD32 (eBioscience) before staining with fluorescence-conjugated antibodies (Supplementary Table S1) in flow cytometry staining buffer with Brilliant Stain Buffer (BD Biosciences). Intracellular staining was performed using the FoxP3/Transcription Factor Staining Buffer Set (eBioscience) and cells were fixed in 3.7% formaldehyde. Counting beads (123Count eBeads; eBioscience) were added to the samples before acquisition on a BD LSRFortessa flow cytometer (BD Biosciences). Panel-specific FMO controls and single color controls were prepared for each experiment. Analysis was carried out using FlowJo (BD Biosciences). As an adjunct to conventional gating, for tumor samples the data was quality controlled using the flowAI plugin in FlowJo followed by T-distributed stochastic neighbor embedding (optSNE) dimensionality reduction and FlowSOM clustering analysis.

Tumors were disaggregated using the gentleMACS™ Mouse Tumor Dissociation kit (Miltenyi Biotec). Tumor-draining lymph nodes (or matching inguinal lymph nodes in non-tumor bearing mice) were dissociated through a 70 µm nylon cell strainer. Cells were stained with a fixable viability dye (Thermo Fisher) and blocked with antibodies against murine CD16/CD32 (eBioscience) before staining with fluorescence-conjugated antibodies (Supplementary Table S1) in flow cytometry staining buffer with Brilliant Stain Buffer (BD Biosciences). Intracellular staining was performed using the FoxP3/Transcription Factor Staining Buffer Set (eBioscience) and cells were fixed in 3.7% formaldehyde. Counting beads (123Count eBeads; eBioscience) were added to the samples before acquisition on a BD LSRFortessa flow cytometer (BD Biosciences). Panel-specific FMO controls and single color controls were prepared for each experiment. Analysis was carried out using FlowJo (BD Biosciences). As an adjunct to conventional gating, for tumor samples the data was quality controlled using the flowAI plugin in FlowJo followed by T-distributed stochastic neighbor embedding (optSNE) dimensionality reduction and FlowSOM clustering analysis.