Liberase dl tl

Breast cancer patient samples and blood from healthy donors were obtained with consent (NHS Lothian, Tissue Request No. 2017/SR865). Within ∼30 min of obtaining the donor blood, blood was isolated via the Ficoll density gradient centrifugation following the manufacturer’s protocol and cryopreserved in 10% DMSO. Cryopreserved blood were thawed rapidly and then incubated for 30 min at 4 degrees with appropriate antibodies (Table S1). Fresh breast tumour biopsies were obtained and processed within 1 h of surgical resection. Tumour tissue was first manually dissected and then chemically dissociated with a cocktail of Liberase DL/TL (Roche) for 30 min and then immunostained with anti-CD45 (Table S1) antibody. Dissociated cells were sorted for live singlet cells with appropriate gating strategy on BD FACSAria II (Fig S1).

Table S1 Antibodies used for sorting and validation.

Table S2 Cluster and donor identity of PBMC cells.

Table S3 Differentially expressed genes between PBMC clusters.

Table S4 Cluster and donor identity of BRCA cells.

Table S5 Differentially expressed genes between breast tumour-infiltrating γδ T subtypes.

Table S6 Differentially expressed genes in γδ T subtypes between all breast tumour-infiltrating immune cells.

For cultured cells or 3D co-culture cells, single-cell suspensions were incubated with 2% human Fc Receptor block (eBioscience, Frankfurt, Germany) in PBS for 20 min on ice. After washing with PBS, cells were stained with conjugated antibodies for 30 min at room temperature in the dark. For tumors, spleen, and ascites from mouse models, single-cell suspensions were prepared by tissue extraction, mashing, digestion with Liberase DL, TL (Roche, USA) and DNase І (Sigma-Aldrich, Germany). The erythrocytes were lysed with ACK lysis buffer (Gibco, Thermo Fisher Scientific), filtered by 40 μm pore size filter (Corning, NY, USA), incubated with 2% human Fc receptor blocking (eBioscience, Frankfurt, Germany), and stained with conjugated antibodies. Isotype IgG control was used in each experiment as reference. Flow cytometry was performed using BD Celesta, BD LSRII, or BD Fortessa (BD Bioscience, US). FACS was performed using BD Aria II or BD Melody (BD Bioscience, US). Data were analyzed using FlowJo software (Tree Star Inc., CA, USA).