Quantifying Immune Cell Populations in Tissue Grafts

Flow cytometry was used to quantify the distribution of different DL cell types in both the graft and perfusate compartments as previously described^{25 (link)} (Table 1). Isolated cells were washed and stained with Live/Dead dye (eBioscience) in PBS and then stained with the following antibodies: anti-CD45 (Biolegend), anti-CD3 (Miltenyi Biotec), anti-CD4 (BD Bioscience), anti-CD8 (eBioscience), anti-CD43 (Biolegend), anti-His48 (eBioscience), anti-CD161 (Biolegend), anti-CD45R (eBioscience). Cell counts (cells/mL) were determined using BD Trucount tubes. Flow cytometric compensation was performed using fluorescent compensation beads, and cells were analyzed using a multiparameter flow cytometer (LSRFortessa BD Biosciences). Analysis of flow results was performed with FlowJo Software (Version 10; FlowJo LLC, Ashland, OR).