Fixation permeabilization kit

RAW264.7 cells were differentiated by treating with 20 ng/mL of interleukin-4 (IL-4) for 24 h to obtain M2 macrophages. Subsequently, 1 mL of cell medium containing M2 macrophages (1 × 10⁶/mL) was dripped onto the device in a dried state, followed by incubating for 12 h. After subjecting the M2 macrophage-loaded devices to various treatments, the devices were incubated with trypsin and washed with fresh cell medium to collect cells. The obtained cells were then incubated with anti-CD11b and anti-CD86 antibodies for 20 min at 4 °C and then washed with PBS. Anti-CD206 antibody was incubated with cells for 60 min on ice, following the use of a fixation/permeabilization kit (BioLegend). All samples were analyzed by flow cytometer (BD Accuri C6) and the data were analyzed using the FlowJo v10.8.1.

Mice tumours were collected, dissociated mechanically, digested with 2 mg/ml Collagenase IV (sigma) and 0.2 mg/ml DNase I (Biofroxx) in serum-free DMEM medium at 37 °C. After 40 min, enzyme activity was neutralized by addition of cold RPMI-1640/10% FBS and tissues were passed through 70 μM cell strainers (Biologix group Limited) and single-cell suspensions in T cell culture medium (RPMI-1640, 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 0.5% β-mercaptoethanol) were stimulated with Cell Activation Cocktail (with Brefeldin A) for 4 h (for intracellular staining). After stimulation, cells were incubated with anti-CD16/CD32 (Biolegend) or Human TruStain FcX™ before staining with fluorochrome-conjugated monoclonal antibodies. Cell surface staining was done for 30 min at 4 °C. Intracellular staining was done using a fixation/permeabilization kit (Biolegend). Zombie Violet Fixable Viability kit (1:400; Biolegend) was added to exclude dead cells. Flow cytometry data analysis was performed by using CytExpert. The general flow cytometry gating strategy was shown in Supplementary Fig. 5a.

After live C. albicans infection, mice were sacrificed at the indicated time after infection and perfused with 1×PBS. Kidneys, spleen and lymph nodes were homogenized in ice cold tissue grinders, filtered through a 70 μm cell strainer and the cells collected by centrifugation at 400 g for 5 min at 4°C. Cells were resuspended and subjected to flow cytometry. Cell surface staining was done for 30 min at 4°C. Intracellular staining was done using a fixation/permeabilization kit (Biolegend). Zombie Violet Fixable Viability kit (1:400; Biolengend) was added to exclude dead cells. Flow cytometry data analysis was performed on CytExpert.