H 2kb af6 88.5

Tumors were first weighed, minced, and treated with collagenase at 37°C for 30 min. Single-cell suspensions were prepared in PBS/1% BSA/0.1% azide (PAB). A total of 10⁶ cells were then treated with Fc block (clone 24G2) and surface-stained at 4°C for 30 min with a mixture of directly conjugated primary Abs specific for CD8 (53–6.7), CD45 (30-F11), IFN-γ (XMG1.2), and H-2K^b (AF6–88.5) (BD Biosciences). All samples were washed in 1 ml of PAB. For measuring intracellular proteins including IFN-γ, cells were first permeabilized using BD Cytofix/Cytoperm (BD Biosciences) solution at 4°C for 15 min and then labeled with anti–IFN-γ Ab. Hypoxia staining was done as previously described (20 (link)). Following surface staining and permeabilization, cells were resuspended in blocking solution containing milk and mouse IgG overnight at 4°C. Cells were then labeled with ELK-3–51-Cy3 for 3 h at 4°C and analyzed using a 12-color LSRII (BD Biosciences) flow cytometer and FlowJo software (Tree Star). Doublet exclusion was done for all experiments based on side and forward scatter. Data are represented as a percentage of CD45⁺ or CD8⁺ events. Fluoroscence minus controls were included in each experiment.

Following red blood cell lysis and blockade of Fc receptors with anti-CD16/32 antibodies, spleen cells were stained with the following directly conjugated antibodies: CD11b (M1/70), CD11c (HL3), CD86 (GL1), I-A/I-E (M5/114.15.2) and H-2K^b (AF6-88.5) (BD Biosciences); CD8α (53-6.7), CD80 (16-10A) and CD19 (eBio1D3) (eBiosciences); CD45.2 (104) CD45.1 (A20), and CD3ε (145-2C11) (BioLegend). Rabbit anti-calreticulin monoclonal antibody and fluorescence-labeled goat-anti-rabbit IgG polyclonal secondary antibody were purchased from Abcam. Fixable viability dyes (Invitrogen) were used to exclude dead cells. Flow cytometry was performed on LSRII or LSRFortessa cytometers (BD Biosciences). Analysis was performed using FlowJo software (Treestar). Fluorescence-activated cell sorting (FACS) was performed using a FACSAria (BD Biosciences).
For DC isolation, spleens were injected with 1 mg/mL collagenase IV (Sigma), 20 µg/mL DNAse I (Roche) were incubated at 37 °C for 15–20 min, and passed through a 70-µm filter to generate single cell suspensions. Cells were stained with CD3ε (145-2C11) and CD19 (eBio1D3) biotinylated antibodies, followed by secondary streptavidin staining to eliminate T and B cells.