Stabilizing fixative

For this study, 50 μL whole blood samples were stained with leukocyte marker antibodies using an established 5-fluorophore-6-marker surface staining method, as previously described [14 (link)]. In brief, whole blood was incubated with a cocktail of monoclonal mouse anti-human antibodies: CD2-APC, CD36-PE, CD16-APC-H7, CD45-AmCyan, CD19-V450 and rat anti-human chemoattractant receptor-homologous molecule expressed on T helper type 2 (Th2) cells (CRTh2)-AF647 (all BD Biosciences). Red blood cells were lysed with BD FACS Lysing Solution before fixation in Stabilizing Fixative and transfer to TruCOUNT Tubes (all BD Biosciences).

Following collection and wash, co-cultures of NK and K-562 cells were incubated with Fixable Viability Dye eFluor™ 506 (1:500) (eBioscience cat #65-0866-14) during the blocking stage. Samples were then incubated with surface stains CD56 (Biolegend cat #318318; RRID : AB_604107), CD11a (Biolegend cat#301207; RRID : AB_10660819), and CD71 (Biolegend cat #334110; RRID : AB_2563117) at 4°C for 30 minutes. K-562 cells were identified as CD56-, CD11a-, CD71+. Samples were fixed using Stabilizing Fixative (BD cat #338036; 1:3 dilution).

The single-cell suspensions obtained after biopsy digestion were labeled with the following antibodies: anti-CD45, anti-CD3, anti-CD11b, and anti-EPCAM, according to the manufacturer’s instructions. Detailed information on the antibodies used in this study can be found in Table 2. Cell viability was assessed using the Zombie Aqua Fixable Viability Kit (BioLegend). The cells were then fixed with the Stabilizing Fixative (BD) before being analyzed using the FACSCanto II flow cytometer (BD).

PBMCs (5 × 10⁵) were stimulated with peptides, DMSO, and positive-control SEB overnight at 37°C (5% humidity), where brefeldin A (Thermo Fisher, Waltham, MA) was added after the first 2 h in culture. After stimulation, viability was determined using a Ghost dye Red 780 (Tonbo Biosciences, San Diego, CA). The cells were surface stained with anti-Asian elephant CD8 and CD4 (custom made at the Monoclonal Antibodies Core Facility at the MD Anderson Cancer Center); the cells were phycoerythrin and Pacific blue labeled, respectively, and then lysed and permeabilized using a Cytofix/Cytoperm kit (BD). The cells were then stained for CD3 (Abcam; clone CD3-12) and IFN-γ (Podiceps; clone AE10F4G11, labeled in-house with fluorescein isothiocyanate). An allophycocyanin-labeled anti-rat secondary was added after incubation with primary antibodies in order to detect bound anti-CD3 primary antibody. The cells were then washed and fixed in stabilizing fixative (BD Biosciences). Cells were acquired on a BD FACSCanto II flow cytometer. At least 100,000 events were collected for each sample, determined by BD FACSDiva software. After acquisition, analysis was performed using FlowJo v10 software (TreeStar).

Whole blood was diluted 1:5 with HEPES saline buffer and incubated with a cocktail of the following fluorescent conjugated antibodies at saturating concentrations: PAC‐1 fluorescein (FITC), CD62P phycoerythrin (PE), CD42b PE‐Cy5, or IgG1Κ PE isotype control (all BD Pharmingen) for exactly 15 min. Six reaction tubes were used that were identical in function and agonist concentrations as those used for MPAs. However, ADP at the concentration used (1.5 μmol/L) caused maximal PAC‐1 binding in all participants, so was not included in statistical analysis. Following incubation, samples were fixed with 800 μL of stabilizing fixative (BD Biosciences) and were then stored at 4°C until analysis by flow cytometry (BD FACSCanto II) within 24 h. Samples were run at a low flow rate until 10,000 platelet positive events were counted. To account for spectral overlap between the three fluorophores, single‐stained compensation beads were used (BD Biosciences). For both MPAs and platelet surface receptor binding, samples were incubated at room temperature with the exception of tubes containing AA and collagen, which were incubated at 37°C using a dry block heater (Ratek DBH20D, Victoria, Australia).