Cytofix cytoperm buf kit

For cell surface staining, cell suspensions were washed twice in PBS and stained with indicated fluorescent labelled antibodies for 30 min on ice and washed with PBS. For TILs isolation, tumours were cut, minced, followed by incubation with Type IV Collagenase(Sigma) and Type I Deoxyribonuclease (Sigma) for 60 min at 37 °C with gentle shaking. After passing through a 70-µm filter, lymphocytes were purified from the interface of mouse Ficoll gradient centrifugation (MultiSciences). For intracellular staining, the cells were sorted for fixation and permeabilization using the Cytofix/CytoPerm buf kit (Cat# 554714, BD Bioscience). For detecting the proliferation of T-cell, human HLA-A2⁺ T lymphocytes were labelled with CFSE, and activated in vitro with anti-CD3 pre-coated U-96-plates in present of 100 U/ml IL2. When needed, irradiated (100 Gy) breast cancer cells were added in co-culture system at the indicated ratio in triples. All flow cytometry analysis were conducted on CytoFlex (Beckman) and the data were analyzed using FlowJo software according to the manufacturers’ instructions. Compensation beads were used to evaluate spectral overlap, compensation was automatically calculated. All antibodies used for flow cytometry analysis are listed in Supplementary Table 3.

The tumors were cut into small pieces, mechanically disrupted and filtered through a 70 µm mesh to generate a single-cell suspension. Dissociated tumor cells were lysed with Red Blood Cell Lysis Solution (No. 420301, Biolegend) and incubated with phorbol 12-myristate 13-acetate (PMA) (No. P1585, Sigma-Aldrich, St. Louis, MO, USA), ionomycin (No. 73724, Stemcell, Vancouver, BC, CAN) and Golgi stop (No. 554715, BD Pharmingen) at 37°C. For cell surface staining, cell suspensions were stained with indicated fluorescent labeled antibodies for 30 min on ice. For intracellular staining, the cells were sorted for fixation and permeabilization using Cytofix/CytoPerm BUF KIT (No. 554714, BD Pharmingen) according to the manufacturer’s guidelines and incubated with primary antibodies. Flow cytometry acquisition was carried out on LSRFortessa (BD Biosciences, San Jose, CA, USA) or a Navios and Gallios (Beckman Coulter, Miami, FL, USA). Compensation beads were used to evaluate spectral overlap. The data were analyzed using FlowJo and CytExpert software according to manufacturers’ instructions.