αcd44 apc

Flow cytometry staining was performed with panels of antibodies specific for activated/memory T cells (αCD3-Biotin, αCD4-FITC, αCD8-APCeF780, αCD62L-PeCy7, αCD44-APC; all from BioLegend) in PBS with 0.5% FCS 2 mM EDTA for 15 min. For intracellular staining cells will be stimulated with a mix containing 10 µg/ml Brefeldin A (Sigma), 50 ng/ml PDBu (Sigma) and 500 ng/ml ionomycin (Sigma) in RPMI-1640 (PAN Biotech) plus 10% FCS (Biochrom) plus 1% penicillin/streptomycin (Lonza) plus 2 mM L-glutamine (Lonza) for 4 h. The cells were then formalin-fixed, permeabilized (0.05% Triton X-100 in PBS) and stained for cytokines (αIL-2-PE, αIFNγ-PeCy7), and transcription factors (αFOXP3-FITC) or perforin (αPerforin-APC) for 1 h. All antibodies were from Biolegend. Cells were analyzed with Gallios and Cytoflex S flow cytometers (Beckman Coulter) and FlowJo Software (Beckton Dickinson).

CD4⁺ or CD8⁺ T‐cells were purified from aged mice using Magnetic‐activated cell sorting (MACs) as described above. T‐cells were stimulated (10⁴–5 × 10⁴ cells/well) in 96‐well flat bottom plates (Millipore Sigma; cat. no. CLS3997) coated with αCD3 (10 µg/ml; BD Biosciences; cat. no. 553057) and αCD28 (2 µg/ml; BD Biosciences; cat. no. 553294) antibodies in RPMI 1640 media supplemented with 10% FBS. On day 3 of culture, T‐cells were harvested, and intracellular cytokine staining was performed as previously described (Henry et al., 2008, 2010) to determine interleukin‐2 (αIL‐2 PE; Biolegend; cat. no. 503808) and interferon‐gamma (αIFN‐γ APC; Biolegend; cat. No. 505810) production using flow cytometry (% positive and mean fluorescence intensity [MFI]). To determine CD44 and PD‐1 surface expression on murine T‐cells, cells were surface stained with αPD‐1 PE (Biolegend; cat. no. 135205) and αCD44 APC (Biolegend; cat. no. 103012) for 1 h (covered on ice) and the MFI for each marker was determined using flow cytometry. All flow cytometry data were analyzed using the FlowJo software (BD Biosciences).

Nanobiotix provided radiation-enhancing nanoparticles (NBTXR3). Bristol Myers Squibb provided αPD1. Antibodies for flow cytometry, including αCD45–Pacific Blue (catalog 103126), αCD4–APC/Fire 750 (catalog 100568), αCD8–PerCP-Cy5.5 (catalog 100734), αCD62L–PE-Cy7 (catalog 104418), and αCD44-APC (catalog 103012) were purchased from BioLegend. Bouin’s fixative solution for staining lung metastases was from Polysciences Inc. (catalog 16045-1).