Lsrfortessa x 20 cell analyzer

T cells from the reovirus-specific T-cell bulk or ex vivo tissues were co-cultured with reovirus-infected target cells (E/T=1:1) or peptides (1 µg/mL). Unless otherwise indicated, the irrelevant cell line TC1 was used as target. Alternatively, serial dilutions of peptides ranging from 10 µM to 10 pM were added to T cells from the reovirus-specific T-cell bulk. When peptides were presented in the context of D1 cells, peptides were incubated with D1 cells for 1 hour before overnight incubation with lipopolysaccharides (LPS, 10 μg/mL). For SLP processing experiments, D1 cells were preincubated for 1 hour with SLPs in concentrations between 10 µM and 1 pM after which LPS (10 µg/mL) was added to each well for an additional 23 hours. Effector cells and target cells, peptides, or peptide-loaded D1 cells were co-cultured for 6 hours in the presence of BD GolgiPlug (BD Biosciences). PMA (20 ng/mL) and ionomycin (1 µg/mL) were used as positive control. After incubation, cells were washed and stained for CD8α (53–6.7; BioLegend). Thereafter, cells were fixed with Fixation Buffer (BioLegend) according to the manufacturer’s instructions, followed by staining for intracellular IFNγ (XMG1.2; BioLegend). After completion of the staining protocol, samples were fixed in 1% paraformaldehyde and acquired using a BD LSRFortessa X20 cell analyzer (BD Biosciences).

Freshly harvested tumors were dissociated to single cell suspensions and stained with fluorochrome-conjugated immune phenotyping antibodies for lymphocyte and myeloid populations for flow cytometry purposes as previously described17 (link) and detailed in online supplemental materials. Fully stained samples were run through a BD LSRFortessa X-20 Cell Analyzer using BD FACSDiva software (BD Biosciences). Post-acquisition analyses were performed using Omiq.ai (https://app.omiq.ai/) following the recommended pipeline (see also online supplemental materials).

Blood samples were obtained on the same day of the operation and just before the start of the operation. For the 15 patients treated with preoperative chemotherapies, blood samples were collected before the start of these treatments. Serum levels of CCL5 were measured using a cytometric bead‐based immunoassay kit according to the manufacturer's protocol (Human RANTES Flex Set; BD Biosciences). Briefly, capture bead populations with distinct fluorescence intensities and coated with RANTES‐specific capture Ab were mixed together in equal volumes, where 50 μL of each sample and 50 μL of phycoerythrin‐conjugated detection Ab was added to 50 μL of a mixed‐bead population. This mixture was incubated for 3 hours at room temperature in the dark to form sandwich complexes. The beads were then washed with wash buffer and analyzed with a BD LSRFortessa X‐20 cell analyzer and FCAP Array software (BD Biosciences).

The BioLegend LEGENDplex Mouse Inflammation Panel (13‐plex) and LEGENDplex Mouse Proinflammatory Chemokine Panel (13‐plex) were used to measure proinflammatory cytokines and chemokines. Assays were performed according to the manufacturer's protocol with modifications in sample preparation. Briefly, lung tissue was homogenized by freeze–thaw cycles and mechanical disruption in ice‐cold assay buffer (10 μL/mg tissue). The supernatant was used to analyze 13 cytokines, including IL‐1α, IL‐1β, IL‐17A, MCP‐1, IFN‐γ, and TNF‐α, simultaneously according to the manufacturer's instructions. Similarly, 13 chemokines, including MCP‐1 (CCL2), RANTES (CCL5), IP‐10 (CXCL10), Eotaxin (CCL11), TARC (CCL17), MIP‐1α (CCL3), MIP‐1β (CCL4), MIP‐3α (CCL20), and KC (CXCL1), were also measured. Data were acquired on the BD LSRFORTESSA X20 cell analyzer equipped with FACSDIVA 9.0 software (BD Biosciences) and analyzed with the LEGENDplex Data Analysis Software Version 8.0 (BioLegend). Results were normalized to lung lysate protein levels measured with the Pierce BCA Protein Assay Kit (Thermo Scientific).