Human naive cd8 t cell isolation kit

Peripheral blood samples were obtained from 27 female and 12 male healthy individuals who did not have a history of autoimmune disease, diabetes mellitus, renal disease, cardiovascular disease or cancer except skin cancer. Individuals with hypertension or hypercholesterinemia were included if controlled on treatment. 19 of these 39 individuals were older than 60 years. In addition, samples were obtained from 128 blood or platelet donors. These samples were deidentified except for whether donors were younger than 35 years or older than 60 years. The studies were approved by the Stanford University Institutional Review Board, and participants gave informed written consent.
Untouched CD4⁺ T cells were purified from peripheral blood or leukapheresis samples of healthy volunteers with a human CD4⁺ T Cell enrichment kit (STEMCELL Technologies), followed by density gradient centrifugation using Lymphoprep (STEMCELL Technologies). Naive CD4⁺ T cells were further isolated by negative selection with anti-CD45RO magnetic beads (Miltenyi Biotec). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Naive CD8⁺ T cell were isolated from PBMCs using a human naive CD8⁺ T cell isolation kit (STEMCELL Technologies). CD14⁺ monocytes were isolated from PBMCs using anti-CD14 magnetic beads (Miltenyi Biotec). Purity of isolated cells was 90% or higher.

CD8 T_RM cells were differentiated in vitro as described previously.^{42 (link)} Briefly, naive CD8⁺ T cells were purified from splenocytes using mouse naive CD8⁺ T cell isolation kit (Stemcell Technologies) then cultured in RPMI-1640 medium (plus β-mercaptoethanol) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% penicillin-streptomycin (cRPMI). 100 ng/mL IL-2 and anti-CD3/CD28 coated Dynabeads (Thermo Fisher) at 1:1 cell to bead ratio were added and cells incubated for 48 h. Then media volume was doubled, each well split into a second well, and 5 ng/mL TGF-β and 100 ng/mL IL-33 were added and incubated for another 48 h before harvest for flow cytometry and RT-qPCR analysis.
For human cells, naive CD8⁺ T cells were purified from PBMCs using human naive CD8⁺ T cell isolation kit (Stemcell Technologies) then cultured in cRPMI and treated as above with the human equivalent Dynabeads and cytokines. FICZ and CH223191 were added at a concentration of 200 nM and 1μM, respectively, as indicated in the text.