CD8+ T cells were purified from the mixed cell suspension using a negative magnetic bead selection kit (Miltenyi Biotec, Auburn, CA) as previously described by us (17 (link), 23 (link)). Dead cells were removed using the Dead cell removal kit (Miltenyi Biotec). To remove stromal fibroblasts from the mixed cell suspension anti-fibroblasts microbeads were also added. Purity of the CD8+ T cell population was higher than 90% following two rounds of negative selection (17 (link), 23 (link)).
To separate CD103-CD8+ T cells from CD103+CD8+ T cells, the purified CD8+ T cell suspension was incubated with CD103-PE antibody (Miltenyi Biotec) for 10 minutes after which anti-PE ultra-pure magnetic beads (Miltenyi Biotec) were added. CD103- were separated by negative selection and CD103+ by positive selection resulting in a purity range between 90-95% for CD103- and CD103+ CD8+ T cells respectively (17 (link), 23 (link)).
To separate CD103-CD8+ T cells from CD103+CD8+ T cells, the purified CD8+ T cell suspension was incubated with CD103-PE antibody (Miltenyi Biotec) for 10 minutes after which anti-PE ultra-pure magnetic beads (Miltenyi Biotec) were added. CD103- were separated by negative selection and CD103+ by positive selection resulting in a purity range between 90-95% for CD103- and CD103+ CD8+ T cells respectively (17 (link), 23 (link)).
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