Krome orange

Flow cytometric analysis of samples was performed as previously described (39 (link), 55 (link)). Briefly, whole blood or PBMCs were stained for 30 minutes at room temperature, and red blood cells lysed (in the case of whole blood) using Red Blood Cell Lysis Buffer (BioLegend). Samples were acquired on an Attune NxT cytometer (Thermo Fisher Scientific) and data analyzed using FlowJo 10.6.2 (BD Biosciences). Absolute cell counts were ascertained by use of counting beads in LUCID DURAclone staining tubes (Beckman Coulter). The gating strategy used is shown in Supplemental Figures 5 and 6.
The following antibodies (clones) were used in this work: CD3 (HIT3a)–FITC, CD14 (M5E2)–PerCP/Cy5.5, CD4 (RPA-T4)–APC/Cy7, CD8 (SK1)–APC, CD56 (5.1H11)–BV711, CD14 (M5E2)–BV650 (BioLegend), CD3 (UCHT1)–FITC, CD4 (13b8.2)–PacificBlue, and CD8 (B9.11)–KromeOrange (Beckman Coulter).

The thrombi obtained after MT were suspended in a container with RPMI 1640 medium 1640 (Gibco™) and stored at 4 to 6 °C for no longer than 24 h until analysis was started in order to avoid decrease in detection of cell surface markers from selected cell populations with different half-life time [18 (link), 19 (link)]. Leukocytes were obtained and analyzed as previously published [20 ]. Briefly, the clots were finely minced and disaggregated to obtain a single cell suspension in 2 to 3 mL of RPMI 1640 using a scalpel. Then, cell suspensions were filtered and washed with phosphate-buffered saline (PBS). Leukocytes were pelleted by centrifugation, resuspended in 100 μL of PBS, and labeled by direct immunofluorescence using CD45 monoclonal antibody conjugated with Krome Orange (Beckman Coulter™). Labeled cells were acquired in a Navios EX™ flow cytometer and were analyzed using Kaluza software (Beckman Coulter™). Finally, leukocytes were classified as granulocytes, monocytes, or lymphocytes according to their side scatter complexity and CD45 level of expression. A threshold of at least 100 leukocytes was set for viable clots. In case there were several thrombi extracted from the same or different vessels, clot obtained in the first pass was selected for analysis from each MT.