Navios ex

ROS levels were analysed using the fluorogenic probe CellROX^® Green Reagent (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). At the specified time points after radiation-based treatments, the CellRox reagent was added in the culture medium to a final concentration of 5 µM. After 30 min incubation in the dark at 37 °C, cells were washed twice with PBS and harvested. Samples were acquired on a flow cytometer (Navios EX, Beckman Coulter, Pasadena, CA, USA) and results were analysed using Kaluza Flow Cytometry Analysis v2.1 software (Beckman Coulter, Pasadena, CA, USA). Relative fluorescence intensities were obtained from the median fluorescence intensity of each histogram of the different experimental conditions.

At least 100,000 cells per sample were acquired using a 3-laser, 10-color flow cytometer (Navios-Ex; Beckman Coulter, Brea, CA, USA) for T-cell receptor analysis or using a 13-color FACS Celesta (BD Bioscience) for immune cell population analyses. The antibodies list and gating strategies are summarized in the Supplementary Table S1. Lymphocytes and myeloid cells were characterized using state-of-the-art markers. Specifically, cells were gated for size, singlets, and then by positive and negative markers: CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells, CD3-CD335⁺ NKs, CD19⁺ B cells, Gr1⁻CD11b⁺CD11c⁺ monocytes, SSC^highCD11b⁺Gr1⁺ granulocytes, CD11c⁺CD11b⁻Gr1⁻ antigen-presenting cells (APC), SSC^lowCD11b⁺Gr1⁺ myeloid-derived suppressor cells (MDSC), and T cells were further analyzed to investigate the exhaustion status (Tim3⁺PD-1⁺).

For the analysis of human lymphocyte subsets, peripheral blood samples were stained with fluorochrome-conjugated or biotinylated Abs (Supplementary Table 6). Cells were analyzed on a Navios EX flow cytometer with Kaluza version 2.1 (Beckman Coulter).
Single-cell suspensions of splenocytes and bone marrow cells from Psmb9^+/+, Psmb9^G156D/+, and Psmb9^G156D/G156D were incubated with an Ab against CD16/32 to block non-specific binding of Abs to Fc receptors. Then, the cells were stained with fluorochrome-conjugated or biotinylated Abs (Supplementary Table 6). Dead cells were excluded by staining with Fixable Viability Dye (eBioscience). To detect MHC class I-mediated presentation of endogenous OVA, splenocytes were stained with an Ab against OVA-derived peptide/H2-K^b complex (Supplementary Table 6). Cells were analyzed on a FACS Verse with FACSuite software v1.0.5.3841 or FACS Aria II with FACSDiva software version 8.0.2 (BD Biosciences) and data were analyzed with FlowJo software version 9.9.6 (BD Bioscience). Representative gating strategies are shown in Supplementary Fig. 9.