E123count beads

Samples of perfusate (4 ml) were collected into EDTA vacutainers, 0.4 ml dimethyl sulfoxide (DMSO) was added and well-mixed; 2 ml of this solution was then transferred into a cryogenic storage vial, moved to a CoolCell® Cell Freezing Container and stored in a −80°C freezer. Immunophenotyping of the human perfusate samples was performed on a BD LSR II flow cytometer (Becton Dickinson, Oxford, United Kingdom). Leukocytes were identified and gated as CD45+ and their viability assessed using an eFluor™ 506 viability dye (eBioscience, California, USA). Following this, a panel of antibodies was utilized to characterize T helper cells (CD3ε+CD4α+), cytotoxic T cells (CD3ε+CD8α+), double-positive T cells (CD3ε+CD4α+CD8α+), double-negative T cells (CD3ε+CD4α-CD8α-), γδ T cells (γδ+), B cells (CD3ε-CD21+), classical monocytes (CD14+CD163-), non-classical monocytes (CD14+CD163+), immature neutrophils (6D10+2B2-), mature neutrophils (6D10+2B2+), mature eosinophils/basophils (6D10-2B2+), and natural killer cells (CD335+). Cells were treated with red blood cell lysing solution (BD Biosciences, United Kingdom), washed, and resuspended in 0.3 ml of staining buffer. A 20 ml quantity of e123count beads (eBioscience, California, USA) was added and samples were analyzed for 3 min. All gating strategies and analysis were performed using FlowJo version 10.0.6 (12 ).