Rat anti il2

Antigen expression was confirmed on ice-cold acetone fixed 8 μm cryostat sections of SKRC52 and CT26-CAIX stained with IL2-XE114-TNF^mut and IL2-F8-TNF^mut (final concentration 5 μg/mL) and detected with rat anti-IL2 (eBioscience 14-7029-85) and anti-rat AlexaFluor488 (Invitrogen A21208). For vascular staining goat anti-CD31 (R&D AF3628) and anti-goat AlexaFluor594 (Invitrogen A11058) antibodies were used. IL2-KSF-TNF^mut (specific for an irrelevant antigen) was used as negative control. Slides were mounted with fluorescent mounting medium and analyzed with Axioskop2 mot plus microscope (Zeiss).
For ex vivo immunofluorescence analysis, mice were injected with 50–60 μg IL2-XE114-TNF^mut, IL2-F8-TNF^mut, or IL2-KSF-TNF^mut and sacrificed 24 h after injection. Organs were excised and embedded in cryo-embedding medium (Thermo Scientific) and cryostat section (10 μm) were stained using the following antibodies: rat anti-IL2 (eBioscience 14-7029-85) and anti-rat AlexaFluor488 (Invitrogen A21208). For vascular staining goat anti-CD31 (R&D AF3628) and anti-goat AlexaFluor594 (Invitrogen A11058) antibodies were used. Slides were mounted with fluorescent mounting medium and analyzed with Axioskop2 mot plus microscope (Zeiss).

SKRC52-hFAP cells were detached with 50 mM EDTA, pH 8.0, in PBS for 10 min at 37 °C. A total of 10 mL of RPMI was used for neutralization and the cells were centrifuged for 5 min at 900 rpm. A wash in 50 mL of a FACS buffer (0.5% BSA, 2 mM EDTA in PBS) was performed and the cells were resuspended at a final concentration of 5 × 10⁶ cells/mL. The cells were passed through a strainer to dissolve clumps and seeded at 500,000 cells of 100 µL into a 96-well U-bottom plate. The cells were incubated for 30 min on ice and then the plate was centrifuged for 3 min at 1500 rpm. Next, a titration of IL2-7NP2-TNF^mut primary antibody was added to the cells in 100 µL of the FACS buffer and incubated for 1 h on ice. Washing from the primary antibody was performed with 100 µL of the FACS buffer, then rat anti-IL2 (eBioscience, Waltham, MA, United States, catalog: 14-7029-85) and anti-rat AlexaFluor488 (Invitrogen A21208) were used for the detection of IL2-7NP2-TNF^mut. Zombie NIR (Biolegend, San Diego, CA, USA) was used as live/dead staining and the data were acquired using a CytoFLEX cytometer (Beckman Coulter, Pasadena, CA, USA). The images were analyzed with FlowJo. The gating strategy is reported in the supplementary material (Supplementary Figure S2).

Antigen expression on SKRC52 cells was confirmed by flow cytometry. Cells were centrifuged and washed in cold FACS buffer (0.5% BSA, 2 mM EDTA in PBS) and stained with IL2-XE114-TNF^mut (final concentration 10 μg/mL) and detected with rat anti-IL2 (eBioscience 14-7029-85) followed by staining with anti-rat AlexaFluor488 (Invitrogen A21208). IL2-KSF-TNF^mut (specific for an irrelevant antigen) was used as negative control.