Easysep na ve cd8 t cell enrichment kit

Naïve CD8 T cells were purified from PBMCs using EasySep naïve CD8 T‐cell enrichment kit (Stem Cell Technology, Vancouver, B.C., Canada). Total RNA was extracted using RNeasy Plus Micro kit (Qiagen, Hilden, Germany) and reversed transcribed using Invitrogen Superscript VILO RT‐PCR Master Mix (Thermo Fisher Scientific). Real‐time quantitative PCR was performed with ABI Prism 7900HT Detection System (Applied Biosystems, Foster City, CA, USA) using Taqman Universal Master Mix II, no UNG (Thermo Fisher Scientific) and Taqman probe sets. Probe sets used are as follows: IL7R (Hs00902334_m1), CCR7 (Hs01013469_m1), SELL (Hs00174151_m1), TCF7 (Hs01556515_m1), and RPLP0 (Hs99999902_m1). All data are presented as relative expression normalized to RPLP0 expression, as this control gene is found to be expressed the most consistently between young and old naïve CD8 T cells.

Pure CD8⁺ T cells were isolated from healthy donor PBMC by using the EasySep naïve CD8⁺ T cell enrichment kit (#19158, StemCell Technologies, Vancouver, BC, Canada). Monocytes-grown dendritic cells were loaded with staphylococcal enterotoxin B (SEB, 1 ng/mL). The previously isolated CD8⁺ T cells were co-cultured with SEB-loaded dendritic cells for six days. On day 7, pure T cells were harvested and adjusted to 10⁶ cells/mL for running the chemotaxis assay [33 (link),47 (link)].