Cd63 h5c6

Cells harvested after overnight incubation were stained following standard protocols. Experiments used antibodies specific for human FcR block (BioLegend #422302), CD11c (Bu15, BioLegend #337214), CD80 (2D10, BioLegend #305220), CD86 (Bu63, BioLegend #374210) HLA-DR (L243, BioLegend #307629), HLA-A,B,C (W6/32, BioLegend #311410), CD14 (M5E2, BioLegend #301828), CD63 (H5C6, BioLegend #353007), CD61 (VI-PL2, BioLegend #336416), CD41/CD61 (PAC-1, BioLegend 362804), CD62p (AK4, BioLegend #304906), mTCRb (H57-597, BioLegend #109206), CD4 (OKT4, BioLegend #317428), CD8 (SK1, BioLegend #344704), and human CD3-APC (Clone OKT3, BioLegend). Color-matched isotype control antibodies were obtained from the same vendors. Flow cytometry was performed with a Stratedigm flow cytometer with electronic gates set on live cells by a combination of forward and side light scatter and 7-AAD (BioLegend), EMA (Invitrogen), Zombie Aqua (BioLegend), or Zombie NIR (BioLegend) exclusion. A minimum of 3 × 10⁴ events were collected per sample, and data were analyzed with FlowJo software (FlowJo LLC).

To analyze the activation of human granulocytes, eosinophils were identified as Siglec8‐positive (7C9; Bio Legend) and CD16‐negative (3G8; Bio Legend) and Annexin V‐negative (120F; IQP). Neutrophils were identified as CD16‐positive (3G8; Bio Legend) and Annexin V‐negative. A total of 50 000 granulocytes were incubated with mAbs for 30 minutes at 4°C, and 10 minutes with Annexin V at 4°C. An assessment of the activation of cell‐surface markers was made by the use of mAbs against the following molecules: CD63 (H5C6; Bio Legend), CD69 (FN50; BD Pharmingen). Cells were washed in PBS containing 0.5% BSA. Data acquisition was done using FACSCanto II (BD Biosciences).